LOCUS       sequence-441135-       14580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EFS promoter driven human codon optimized SpdCas9 directly
            fused to Dnmt3A/3L variant V1 (R887E) 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-V1
FEATURES             Location/Qualifiers
     source          1..14580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    431..1019
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(484..504)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(902..913)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1091..1324
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(1350..1367)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1356..1580
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1626..2054
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1713..1732)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1923..1944
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2063..2083)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2068..2397
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2248..2383
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2310..2329
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2445..2492
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2511..2885
                     /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    3015..3136
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3052..3071)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3106..3125
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3185..3201)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3185..3201)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3198..3220)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3209..3225
                     /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(3233..3239,3240..3257,3258..3263))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3278..3299
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3416..3433)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3587..4175)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3667..3686)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4346..5137,5138..5206))
                     /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     4969..4988
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5207..5311)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5386..5405)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5577..5956
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5958..6156
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6111..6131
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             6174..6354
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6401..6526
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7019..7252
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7437..7481
                     /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    7779..7896
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        8028..8103
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        8165..8376
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      8390..8399
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(8396..8422,8423..8425,8426..10912,10913..10915,
                     10916..12499)
                     /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             8396..12499
                     /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             12500..12547
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             14195..14218
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             14228..14284
                     /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             join(14285..14580,1..415)
                     /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(14430..14448)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
ORIGIN
        1 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
       61 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
      121 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
      181 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
      241 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
      301 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
      361 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtagtgtcg
      421 ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
      481 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
      541 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
      601 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
      661 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
      721 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
      781 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
      841 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
      901 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
      961 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg
     1021 tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     1081 aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt
     1141 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     1201 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     1261 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     1321 agcagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     1381 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     1441 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     1501 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     1561 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     1621 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1681 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1741 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1801 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     1861 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     1921 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     1981 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2041 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2101 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2161 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2221 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2281 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     2341 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     2401 ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat
     2461 cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt
     2521 tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga
     2581 ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg
     2641 acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg
     2701 cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca
     2761 cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc
     2821 gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg
     2881 actgacacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     2941 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     3001 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     3061 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     3121 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     3181 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     3241 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     3301 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     3361 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     3421 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3481 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3541 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3601 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3661 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3721 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3781 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3841 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3901 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3961 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4021 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4081 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4141 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4201 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4261 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4321 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4381 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4441 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     4501 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4561 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4621 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     4681 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4741 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4801 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4861 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4921 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4981 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5041 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5101 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5161 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5221 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5281 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     5341 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     5401 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     5461 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
     5521 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
     5581 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     5641 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     5701 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     5761 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     5821 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     5881 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     5941 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     6001 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     6061 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     6121 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc
     6181 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     6241 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     6301 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     6361 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     6421 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     6481 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     6541 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     6601 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     6661 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     6721 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     6781 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     6841 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga
     6901 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag
     6961 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag
     7021 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga
     7081 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc
     7141 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc
     7201 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt
     7261 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta
     7321 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta
     7381 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga
     7441 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa
     7501 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa
     7561 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat
     7621 cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag
     7681 aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg
     7741 cgccaattct gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt
     7801 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     7861 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga
     7921 gatccagttt ggttaattaa ggtaccgata tcactagtgt tccattaact gcgctaaaaa
     7981 taatttttaa atctttttta gcttcttgct cttttttgta cgtctctgtt ttagagctag
     8041 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     8101 tgcttttttg aattcgctag ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt
     8161 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
     8221 tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
     8281 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
     8341 gttctttttc gcaacgggtt tgccgccaga acacaggact ggttctagag ccaccatgga
     8401 caagaagtac agcatcggcc tggccatcgg caccaactct gtgggctggg ccgtgatcac
     8461 cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag
     8521 catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac
     8581 ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct
     8641 gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga
     8701 agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat
     8761 cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact
     8821 ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat
     8881 caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga
     8941 caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa
     9001 cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct
     9061 ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat
     9121 tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc
     9181 caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat
     9241 cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct
     9301 gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat
     9361 caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca
     9421 gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta
     9481 cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa
     9541 gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca
     9601 gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat
     9661 tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa
     9721 gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt
     9781 cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt
     9841 ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct
     9901 gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa
     9961 cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg
    10021 cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa
    10081 gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg
    10141 cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa
    10201 ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac
    10261 cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct
    10321 gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct
    10381 gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt
    10441 cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct
    10501 gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga
    10561 gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa
    10621 ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga
    10681 aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa
    10741 gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga
    10801 aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat
    10861 gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg acgccatcgt
    10921 gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa
    10981 gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta
    11041 ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa
    11101 ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt
    11161 ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa
    11221 gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct
    11281 ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca
    11341 ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc
    11401 taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat
    11461 cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat
    11521 catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct
    11581 gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac
    11641 cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac
    11701 aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag
    11761 aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc
    11821 tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga
    11881 gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct
    11941 ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc
    12001 cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa
    12061 gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta
    12121 tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca
    12181 caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct
    12241 ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat
    12301 cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc
    12361 cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt
    12421 gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct
    12481 gtctcagctg ggaggcgaca agcgacctgc cgccacaaag aaggctggac aggctaagaa
    12541 gaagaaagga agcatgaacc atgaccagga atttgacccc ccaaaggttt acccacctgt
    12601 gccagctgag aagaggaagc ccatccgcgt gctgtctctc tttgatggga ttgctacagg
    12661 gctcctggtg ctgaaggacc tgggcatcca agtggaccgc tacattgcct ccgaggtgtg
    12721 tgaggactcc atcacggtgg gcatggtgcg gcaccaggga aagatcatgt acgtcgggga
    12781 cgtccgcagc gtcacacaga agcatatcca ggagtggggc ccattcgacc tggtgattgg
    12841 aggcagtccc tgcaatgacc tctccattgt caaccctgcc cgcaagggac tttatgaggg
    12901 tactggccgc ctcttctttg agttctaccg cctcctgcat gatgcgcggc ccaaggaggg
    12961 agatgatcgc cccttcttct ggctctttga gaatgtggtg gccatgggcg ttagtgacaa
    13021 gagggacatc tcgcgatttc ttgagtctaa ccccgtgatg attgacgcca aagaagtgtc
    13081 tgctgcacac agggcccgtt acttctgggg taaccttcct ggcatgaaca ggcctttggc
    13141 atccactgtg aatgataagc tggagctgca agagtgtctg gagcacggca gaatagccaa
    13201 gttcagcaaa gtgaggacca ttaccaccag gtcaaactct ataaagcagg gcaaagacca
    13261 gcatttcccc gtcttcatga acgagaagga ggacatcctg tggtgcactg aaatggaaag
    13321 ggtgtttggc ttccccgtcc actacacaga cgtctccaac atgagccgct tggcgaggca
    13381 ggaactgctg ggccgatcgt ggagcgtgcc ggtcatccgc cacctcttcg ctccgctgaa
    13441 ggaatatttt gcttgtgtgt ctagcggcaa tagtaacgct aacagccgcg ggccgagctt
    13501 cagcagcggc ctggtgccgt taagcttgcg cggcagccat atgggcccta tggagatata
    13561 caagacagtg tctgcatgga agagacagcc agtgcgggta ctgagccttt ttagaaatat
    13621 tgataaagta ctaaagagtt tgggcttttt agaaagcggt tctggttctg ggggaggaac
    13681 gctgaagtac gtggaagatg tcacaaatgt cgtgaggaga gacgtggaga aatggggccc
    13741 ctttgacctg gtgtacggct cgacgcagcc cctaggcagc tcctgtgatc gctgtcccgg
    13801 ctggtacatg ttccagttcc accggatcct gcagtatgcg ctgcctcgcc aggagagtca
    13861 gcggcccttc ttctggatat tcatggacaa tctgctgctg actgaggatg accaagagac
    13921 aactacccgc ttccttcaga cagaggctgt gaccctccag gatgtccgtg gcagggacta
    13981 ccagaatgct atgcgggtgt ggagcaacat tccagggctg aagagcaagc atgcgcccct
    14041 gaccccaaag gaagaagagt atctgcaagc ccaagtcaga agcaggagca agctggacgc
    14101 cccgaaagtt gacctcctgg tgaagaactg ccttctcccg ctgagagagt acttcaagta
    14161 tttttctcaa aactcacttc ctcttggaag cgctgattac aaagacgatg acgataaggg
    14221 atccggcgca acaaacttct ctctgctgaa acaagccgga gatgtcgaag agaatcctgg
    14281 accgatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
    14341 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
    14401 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
    14461 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
    14521 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
//
