LOCUS       sequence-441220-       14580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EFS promoter driven human codon optimized SpdCas9 directly
            fused to Dnmt3A/3L variant cat-Delta (C706A and R832E) 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-cat-Delta
FEATURES             Location/Qualifiers
     source          1..14580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      15..443
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(102..121)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     312..333
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(452..472)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        457..786
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      637..772
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     699..718
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        834..881
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             900..1274
                     /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    1404..1525
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1441..1460)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1495..1514
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1574..1590)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1574..1590)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1587..1609)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1598..1614
                     /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(1622..1628,1629..1646,1647..1652))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1667..1688
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1805..1822)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1976..2564)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2056..2075)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2735..3526,3527..3595))
                     /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     3358..3377
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3596..3700)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3775..3794)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3966..4345
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4347..4545
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4500..4520
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4563..4743
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4790..4915
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5408..5641
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5826..5870
                     /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    6168..6285
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        6417..6492
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6554..6765
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      6779..6788
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6785..6811,6812..6814,6815..9301,9302..9304,
                     9305..10888)
                     /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             6785..10888
                     /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             10889..10936
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             12584..12607
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             12617..12673
                     /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             12674..13384
                     /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(12819..12837)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     13087..13106
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    13400..13988
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(13453..13473)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(13871..13882)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             14060..14293
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(14319..14336)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    14325..14549
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
       61 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
      121 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
      181 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
      241 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
      301 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
      361 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
      421 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
      481 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
      541 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
      601 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
      661 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
      721 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
      781 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgttgaca
      841 attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca
      901 tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg
      961 agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg
     1021 tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca
     1081 acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg
     1141 tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc
     1201 cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg
     1261 aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt
     1321 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     1381 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     1441 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     1501 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     1561 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     1621 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     1681 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     1741 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     1801 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     1861 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     1921 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     1981 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2041 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2101 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     2161 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     2221 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     2281 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     2341 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     2401 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     2461 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     2521 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     2581 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     2641 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     2701 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     2761 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     2821 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     2881 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     2941 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3001 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     3061 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     3121 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     3181 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     3241 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     3301 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     3361 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     3421 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     3481 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     3541 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     3601 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     3661 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     3721 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
     3781 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     3841 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     3901 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
     3961 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
     4021 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     4081 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     4141 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     4201 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     4261 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     4321 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
     4381 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     4441 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
     4501 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagcgcgt tttgcctgta
     4561 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     4621 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     4681 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     4741 gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg
     4801 caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac
     4861 gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat
     4921 taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa
     4981 aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa
     5041 tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc
     5101 ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg
     5161 tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga
     5221 gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag
     5281 gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac
     5341 cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag
     5401 tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag
     5461 cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga
     5521 acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca
     5581 tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc
     5641 tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta
     5701 gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca
     5761 gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc
     5821 aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt
     5881 ttaacataac aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg
     5941 taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt
     6001 caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa
     6061 tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg aacggatcgg
     6121 cactgcgtgc gccaattctg cagacaaatg gcagtattca tccacaattt taaaagaaaa
     6181 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     6241 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     6301 gacagcagag atccagtttg gttaattaag gtaccgatat cactagtgtt ccattaactg
     6361 cgctaaaaat aatttttaaa tcttttttag cttcttgctc ttttttgtac gtctctgttt
     6421 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     6481 ccgagtcggt gcttttttga attcgctagc taggtcttga aaggagtggg aattggctcc
     6541 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
     6601 gtcggcaatt gatccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
     6661 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
     6721 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggactg gttctagagc
     6781 caccatggac aagaagtaca gcatcggcct ggccatcggc accaactctg tgggctgggc
     6841 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga
     6901 ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc
     6961 cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat
     7021 ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca
     7081 cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt
     7141 cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag
     7201 aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc
     7261 ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag
     7321 cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa
     7381 ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag
     7441 cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg
     7501 aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc
     7561 cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct
     7621 ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc
     7681 catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc
     7741 ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt
     7801 gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta
     7861 cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat
     7921 cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct
     7981 gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct
     8041 gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa
     8101 gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa
     8161 cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga
     8221 ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga
     8281 taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac
     8341 cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt
     8401 cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt
     8461 gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga
     8521 aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa
     8581 aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat
     8641 cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta
     8701 tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg
     8761 gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat
     8821 cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga
     8881 cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag
     8941 cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca
     9001 gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat
     9061 cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga
     9121 gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca
     9181 ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg
     9241 gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga
     9301 cgccatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag
     9361 aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat
     9421 gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa
     9481 tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag
     9541 acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat
     9601 gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa
     9661 gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa
     9721 caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa
     9781 aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg
     9841 gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta
     9901 cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa
     9961 gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga
    10021 ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga
    10081 ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct
    10141 gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt
    10201 ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag
    10261 tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat
    10321 cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc
    10381 taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga
    10441 actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc
    10501 cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt
    10561 ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag
    10621 agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga
    10681 taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg
    10741 agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac
    10801 caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg
    10861 gatcgacctg tctcagctgg gaggcgacaa gcgacctgcc gccacaaaga aggctggaca
    10921 ggctaagaag aagaaaggaa gcatgaacca tgaccaggaa tttgaccccc caaaggttta
    10981 cccacctgtg ccagctgaga agaggaagcc catccgcgtg ctgtctctct ttgatgggat
    11041 tgctacaggg ctcctggtgc tgaaggacct gggcatccaa gtggaccgct acattgcctc
    11101 cgaggtgtgt gaggactcca tcacggtggg catggtgcgg caccagggaa agatcatgta
    11161 cgtcggggac gtccgcagcg tcacacagaa gcatatccag gagtggggcc cattcgacct
    11221 ggtgattgga ggcagtcccg caaatgacct ctccattgtc aaccctgccc gcaagggact
    11281 ttatgagggt actggccgcc tcttctttga gttctaccgc ctcctgcatg atgcgcggcc
    11341 caaggaggga gatgatcgcc ccttcttctg gctctttgag aatgtggtgg ccatgggcgt
    11401 tagtgacaag agggacatct cgcgatttct tgagtctaac cccgtgatga ttgacgccaa
    11461 agaagtgtct gctgcacaca gggcccgtta cttctggggt aaccttcctg gcatgaacag
    11521 gcctttggca tccactgtga atgataagct ggagctgcaa gagtgtctgg agcacggcag
    11581 aatagccaag ttcagcaaag tgaggaccat taccaccgaa tcaaactcta taaagcaggg
    11641 caaagaccag catttccccg tcttcatgaa cgagaaggag gacatcctgt ggtgcactga
    11701 aatggaaagg gtgtttggct tccccgtcca ctacacagac gtctccaaca tgagccgctt
    11761 ggcgaggcag agactgctgg gccgatcgtg gagcgtgccg gtcatccgcc acctcttcgc
    11821 tccgctgaag gaatattttg cttgtgtgtc tagcggcaat agtaacgcta acagccgcgg
    11881 gccgagcttc agcagcggcc tggtgccgtt aagcttgcgc ggcagccata tgggccctat
    11941 ggagatatac aagacagtgt ctgcatggaa gagacagcca gtgcgggtac tgagcctttt
    12001 tagaaatatt gataaagtac taaagagttt gggcttttta gaaagcggtt ctggttctgg
    12061 gggaggaacg ctgaagtacg tggaagatgt cacaaatgtc gtgaggagag acgtggagaa
    12121 atggggcccc tttgacctgg tgtacggctc gacgcagccc ctaggcagct cctgtgatcg
    12181 ctgtcccggc tggtacatgt tccagttcca ccggatcctg cagtatgcgc tgcctcgcca
    12241 ggagagtcag cggcccttct tctggatatt catggacaat ctgctgctga ctgaggatga
    12301 ccaagagaca actacccgct tccttcagac agaggctgtg accctccagg atgtccgtgg
    12361 cagggactac cagaatgcta tgcgggtgtg gagcaacatt ccagggctga agagcaagca
    12421 tgcgcccctg accccaaagg aagaagagta tctgcaagcc caagtcagaa gcaggagcaa
    12481 gctggacgcc ccgaaagttg acctcctggt gaagaactgc cttctcccgc tgagagagta
    12541 cttcaagtat ttttctcaaa actcacttcc tcttgctgga tccgattaca aagacgatga
    12601 cgataaggga tccggcgcaa caaacttctc tctgctgaaa caagccggag atgtcgaaga
    12661 gaatcctgga ccgatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
    12721 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
    12781 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
    12841 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
    12901 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
    12961 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
    13021 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
    13081 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
    13141 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
    13201 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
    13261 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
    13321 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
    13381 gtagtgtcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg
    13441 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
    13501 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
    13561 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
    13621 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
    13681 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
    13741 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
    13801 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
    13861 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
    13921 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
    13981 ctccccgcgt cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt
    14041 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt
    14101 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
    14161 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
    14221 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
    14281 aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
    14341 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
    14401 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
    14461 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
    14521 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
//
