LOCUS       sequence-441137-       14580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EFS promoter driven human codon optimized SpdCas9 directly
            fused to Dnmt3A/3L variant V3 (R887E and E814G) followed by 
            P2A-mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14580)
  AUTHORS   Guerra-Resendez RS, Lydon SL, Ma AJ, Bedford GC, Reed DR, Kim S, 
            Teran ER, Nishiguchi T, Escobar M, DiNardo AR, Hilton IB
  TITLE     Characterization of Rationally Designed CRISPR/Cas9-Based DNA 
            Methyltransferases with Distinct Methyltransferase and Gene 
            Silencing Activities in Human Cell Lines and Primary Human T Cells.
  JOURNAL   ACS Synth Biol. 2025 Feb 3. doi: 10.1021/acssynbio.4c00569.
  PUBMED    39898483
REFERENCE   2  (bases 1 to 14580)
  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: "ACS Synth 
            Biol. 2025 Feb 3. doi: 10.1021/acssynbio.4c00569."
COMMENT     Sequence Label: EFS-SpdCas9-Dnmt3A/3L-V3
FEATURES             Location/Qualifiers
     source          1..14580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        135..182
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             201..575
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    705..826
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(742..761)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     796..815
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(875..891)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(875..891)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(888..910)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    899..915
                     /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(923..929,930..947,948..953))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    968..989
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1106..1123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1277..1865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1357..1376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2036..2827,2828..2896))
                     /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     2659..2678
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2897..3001)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3076..3095)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3267..3646
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3648..3846
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3801..3821
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             3864..4044
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4091..4216
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4709..4942
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5127..5171
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    5469..5586
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        5718..5793
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        5855..6066
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      6080..6089
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6086..6112,6113..6115,6116..8602,8603..8605,
                     8606..10189)
                     /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             6086..10189
                     /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             10190..10237
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             11885..11908
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             11918..11974
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             11975..12685
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(12120..12138)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     12388..12407
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    12701..13289
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(12754..12774)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(13172..13183)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             13361..13594
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(13620..13637)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    13626..13850
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      13896..14324
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(13983..14002)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     14193..14214
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(14333..14353)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(14338..14580,1..87)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(14518..14580,1..73)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     join(14580,1..19)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
       61 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
      121 tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg
      181 acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc
      241 gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg
      301 tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc
      361 aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc
      421 tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca
      481 tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca
      541 actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca
      601 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
      661 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
      721 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
      781 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
      841 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
      901 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
      961 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1021 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1081 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1141 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1201 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1261 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1321 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1381 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     1441 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1501 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     1561 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     1621 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     1681 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     1741 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1801 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1861 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     1921 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     1981 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2041 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2101 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     2161 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     2221 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     2281 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     2341 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     2401 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     2461 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     2521 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     2581 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     2641 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     2701 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     2761 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     2821 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     2881 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     2941 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3001 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     3061 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     3121 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     3181 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     3241 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
     3301 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     3361 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     3421 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     3481 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     3541 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     3601 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     3661 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     3721 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     3781 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     3841 aagcagcgcg ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc
     3901 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     3961 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     4021 agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa
     4081 accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga
     4141 ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga
     4201 tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg
     4261 gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga
     4321 gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat
     4381 actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa
     4441 tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc
     4501 tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc
     4561 tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat
     4621 ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg
     4681 tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag
     4741 caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt
     4801 ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt
     4861 tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat
     4921 acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca
     4981 ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca
     5041 cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
     5101 ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat
     5161 gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca
     5221 taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga
     5281 atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg
     5341 gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca
     5401 ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc
     5461 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     5521 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     5581 aattttcggg tttattacag ggacagcaga gatccagttt ggttaattaa ggtaccgata
     5641 tcactagtgt tccattaact gcgctaaaaa taatttttaa atctttttta gcttcttgct
     5701 cttttttgta cgtctctgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     5761 tatcaacttg aaaaagtggc accgagtcgg tgcttttttg aattcgctag ctaggtcttg
     5821 aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc
     5881 cccgagaagt tggggggagg ggtcggcaat tgatccggtg cctagagaag gtggcgcggg
     5941 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
     6001 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
     6061 acacaggact ggttctagag ccaccatgga caagaagtac agcatcggcc tggccatcgg
     6121 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     6181 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     6241 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     6301 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     6361 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     6421 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     6481 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     6541 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     6601 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     6661 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     6721 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     6781 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt
     6841 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     6901 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     6961 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     7021 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     7081 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     7141 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     7201 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     7261 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     7321 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     7381 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     7441 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     7501 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     7561 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     7621 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     7681 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     7741 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     7801 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     7861 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     7921 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     7981 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     8041 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     8101 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     8161 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     8221 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     8281 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     8341 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     8401 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     8461 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     8521 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     8581 gctgtccgac tacgatgtgg acgccatcgt gcctcagagc tttctgaagg acgactccat
     8641 cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc
     8701 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     8761 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     8821 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     8881 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     8941 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     9001 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     9061 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     9121 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     9181 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     9241 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     9301 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     9361 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     9421 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     9481 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     9541 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     9601 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     9661 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     9721 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     9781 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     9841 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     9901 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     9961 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
    10021 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
    10081 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
    10141 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca agcgacctgc
    10201 cgccacaaag aaggctggac aggctaagaa gaagaaagga agcatgaacc atgaccagga
    10261 atttgacccc ccaaaggttt acccacctgt gccagctgag aagaggaagc ccatccgcgt
    10321 gctgtctctc tttgatggga ttgctacagg gctcctggtg ctgaaggacc tgggcatcca
    10381 agtggaccgc tacattgcct ccgaggtgtg tgaggactcc atcacggtgg gcatggtgcg
    10441 gcaccaggga aagatcatgt acgtcgggga cgtccgcagc gtcacacaga agcatatcca
    10501 ggagtggggc ccattcgacc tggtgattgg aggcagtccc tgcaatgacc tctccattgt
    10561 caaccctgcc cgcaagggac tttatgaggg tactggccgc ctcttctttg agttctaccg
    10621 cctcctgcat gatgcgcggc ccaaggaggg agatgatcgc cccttcttct ggctctttga
    10681 gaatgtggtg gccatgggcg ttagtgacaa gagggacatc tcgcgatttc ttgagtctaa
    10741 ccccgtgatg attgacgcca aagaagtgtc tgctgcacac agggcccgtt acttctgggg
    10801 taaccttcct ggcatgaaca ggcctttggc atccactgtg aatgataagc tgggactgca
    10861 agagtgtctg gagcacggca gaatagccaa gttcagcaaa gtgaggacca ttaccaccag
    10921 gtcaaactct ataaagcagg gcaaagacca gcatttcccc gtcttcatga acgagaagga
    10981 ggacatcctg tggtgcactg aaatggaaag ggtgtttggc ttccccgtcc actacacaga
    11041 cgtctccaac atgagccgct tggcgaggca ggaactgctg ggccgatcgt ggagcgtgcc
    11101 ggtcatccgc cacctcttcg ctccgctgaa ggaatatttt gcttgtgtgt ctagcggcaa
    11161 tagtaacgct aacagccgcg ggccgagctt cagcagcggc ctggtgccgt taagcttgcg
    11221 cggcagccat atgggcccta tggagatata caagacagtg tctgcatgga agagacagcc
    11281 agtgcgggta ctgagccttt ttagaaatat tgataaagta ctaaagagtt tgggcttttt
    11341 agaaagcggt tctggttctg ggggaggaac gctgaagtac gtggaagatg tcacaaatgt
    11401 cgtgaggaga gacgtggaga aatggggccc ctttgacctg gtgtacggct cgacgcagcc
    11461 cctaggcagc tcctgtgatc gctgtcccgg ctggtacatg ttccagttcc accggatcct
    11521 gcagtatgcg ctgcctcgcc aggagagtca gcggcccttc ttctggatat tcatggacaa
    11581 tctgctgctg actgaggatg accaagagac aactacccgc ttccttcaga cagaggctgt
    11641 gaccctccag gatgtccgtg gcagggacta ccagaatgct atgcgggtgt ggagcaacat
    11701 tccagggctg aagagcaagc atgcgcccct gaccccaaag gaagaagagt atctgcaagc
    11761 ccaagtcaga agcaggagca agctggacgc cccgaaagtt gacctcctgg tgaagaactg
    11821 ccttctcccg ctgagagagt acttcaagta tttttctcaa aactcacttc ctcttggaag
    11881 cgctgattac aaagacgatg acgataaggg atccggcgca acaaacttct ctctgctgaa
    11941 acaagccgga gatgtcgaag agaatcctgg accgatggtg agcaagggcg aggaggataa
    12001 catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg
    12061 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
    12121 caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca
    12181 gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
    12241 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
    12301 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
    12361 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
    12421 ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag
    12481 gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa
    12541 gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca
    12601 caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg
    12661 cggcatggac gagctgtaca agtagtgtcg ttaagtcgac aatcaacctc tggattacaa
    12721 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
    12781 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
    12841 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
    12901 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
    12961 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
    13021 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
    13081 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
    13141 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
    13201 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
    13261 tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac ttacaaggca
    13321 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
    13381 caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga
    13441 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
    13501 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
    13561 agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc gctgatcagc
    13621 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
    13681 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
    13741 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
    13801 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
    13861 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
    13921 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
    13981 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
    14041 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
    14101 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
    14161 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
    14221 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
    14281 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
    14341 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
    14401 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
    14461 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
    14521 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
//
