LOCUS       sequence-461177-        8339 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express nCas9 nickase (D10) with GFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8339)
  AUTHORS   Chen XD, Chen Z, Wythes G, Zhang Y, Orr BC, Sun G, Chao YK, Navarro 
            Torres A, Thao K, Vallurupalli M, Sun J, Borji M, Tkacik E, Chen H, 
            Bernstein BE, Chen F
  TITLE     Helicase-assisted continuous editing for programmable mutagenesis of
            endogenous genomes.
  JOURNAL   Science. 2024 Oct 11;386(6718):eadn5876. doi: 
            10.1126/science.adn5876. Epub 2024 Oct 11.
  PUBMED    39388570
REFERENCE   2  (bases 1 to 8339)
  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: "Science."; 
            date: "2024-10-11"; volume: "386(6718):eadn5876. doi"; pages: " 
            10.1126/science.adn5876. Epub 2024 Oct 11"
COMMENT     Sequence Label: pCMV-SpCas9(D10A)-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..8339
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      384..393
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(390..416,417..419,420..4493)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             390..4493
                     /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
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4506..4526
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4536..4562
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             4572..4628
                     /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(4629..4631,4632..4634,4635..5348)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4629..4631,4632..4634,4635..5345)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4935..4954)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5282..5303
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5354..5371
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5394..5411)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5400..5624
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5695..5711)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5695..5711)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5708..5730)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5719..5735
                     /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(5743..5749,5750..5767,5768..5773))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5788..5809
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5926..5943)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6097..6685)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6177..6196)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6856..7647,7648..7716))
                     /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     7479..7498
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7717..7821)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7900..7919)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8091..8339,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgcgaga gccgccacca tggacaagaa gtacagcatc ggcctggcca
      421 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
      481 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
      541 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
      601 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
      661 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
      721 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
      781 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
      841 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
      901 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
      961 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     1021 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     1081 gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca
     1141 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     1201 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     1261 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     1321 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     1381 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     1441 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     1501 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     1561 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     1621 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     1681 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     1741 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     1801 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     1861 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     1921 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     1981 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     2041 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     2101 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     2161 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     2221 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     2281 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     2341 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     2401 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     2461 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     2521 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     2581 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     2641 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     2701 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     2761 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     2821 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     2881 accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact
     2941 ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc
     3001 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     3061 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     3121 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     3181 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc
     3241 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     3301 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctaaacg
     3361 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     3421 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     3481 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     3541 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
     3601 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
     3661 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     3721 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     3781 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     3841 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     3901 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     3961 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     4021 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     4081 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     4141 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     4201 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     4261 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     4321 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     4381 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     4441 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggt gactctggtg
     4501 gttctcccaa gaagaagagg aaagtcggct ctggccctgc cgctaagaga gtgaagctgg
     4561 acggatccgg cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc gaagagaatc
     4621 ctggaccgat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     4681 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     4741 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     4801 ggcccaccct cgtgaccacc ctgacctatg gagtgcagtg cttcagccgc taccccgacc
     4861 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     4921 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     4981 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     5041 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     5101 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     5161 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     5221 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     5281 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     5341 acaagtaacc ggtcatcatc accatcacca ttgagtttaa acccgctgat cagcctcgac
     5401 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     5461 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     5521 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     5581 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     5641 aaccagctgg ggctcgatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     5701 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5761 cataaagtgt aaagcctagg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5821 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5881 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5941 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     6001 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     6061 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     6121 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     6181 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     6241 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     6301 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6361 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6421 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6481 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     6541 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6601 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6661 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6721 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6781 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6841 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6901 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6961 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     7021 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     7081 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     7141 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     7201 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     7261 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     7321 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7381 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7441 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7501 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7561 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7621 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7681 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7741 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7801 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     7861 gggagatcga tctcccgatc ccctagggtc gactctcagt acaatctgct ctgatgccgc
     7921 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     7981 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
     8041 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
     8101 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     8161 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     8221 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     8281 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatc
//
