LOCUS       sequence-336052-        8813 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of SpCas9-N57 fusion protein to enhace site-specific DNA 
            integration..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8813)
  AUTHORS   Ma S, Wang X, Hu Y, Lv J, Liu C, Liao K, Guo X, Wang D, Lin Y, Rong 
            Z
  TITLE     Enhancing site-specific DNA integration by a Cas9 nuclease fused 
            with a DNA donor-binding domain.
  JOURNAL   Nucleic Acids Res. 2020 Oct 9;48(18):10590-10601. doi: 
            10.1093/nar/gkaa779.
  PUBMED    32986839
REFERENCE   2  (bases 1 to 8813)
  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"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2020-10-9- 9"; volume: "48"; issue: 
            "18"; pages: "10590-10601"
COMMENT     Sequence Label: pCAG-spCas9-N57
FEATURES             Location/Qualifiers
     source          1..8813
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          610..1626
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1550..1572
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1634..1653
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1705..1770
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1777..1797
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1819..5922
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1819..5922
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1819..5922
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5935..5955
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    6297..6418
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6334..6353)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6388..6407
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6467..6483)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6467..6483)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6480..6502)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6491..6507
                     /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(6515..6521,6522..6539,6540..6545))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6560..6581
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6698..6715)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6869..7457)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6949..6968)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7628..8419,8420..8488))
                     /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     8251..8270
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8489..8593)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8672..8691)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      421 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      481 tgggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc
      541 cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg
      601 cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg
      661 cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc
      721 ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg
      781 ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg
      841 ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc
      901 tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc
      961 gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg
     1021 cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc
     1081 cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt
     1141 acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg
     1201 ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg
     1261 agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc
     1321 gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc
     1381 cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg
     1441 ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg
     1501 ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc
     1561 tggcgtgtga ccggcggctc tagcgcctct gctaaccatg ttcatgcctt cttctttttc
     1621 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc
     1681 tctagagata tcgctagcac catggactac aaagaccatg acggtgatta taaagatcat
     1741 gacatcgatt acaaggatga cgatgacaag atggccccca agaagaagag gaaggtgggc
     1801 attcaccgcg gggtacccat ggacaagaag tactccattg ggctcgatat cggcacaaac
     1861 agcgtcggct gggccgtcat tacggacgag tacaaggtgc cgagcaaaaa attcaaagtt
     1921 ctgggcaata ccgatcgcca cagcataaag aagaacctca ttggcgccct cctgttcgac
     1981 tccggggaga cggccgaagc cacgcggctc aaaagaacag cacggcgcag atatacccgc
     2041 agaaagaatc ggatctgcta cctgcaggag atctttagta atgagatggc taaggtggat
     2101 gactctttct tccataggct ggaggagtcc tttttggtgg aggaggataa aaagcacgag
     2161 cgccacccaa tctttggcaa tatcgtggac gaggtggcgt accatgaaaa gtacccaacc
     2221 atatatcatc tgaggaagaa gcttgtagac agtactgata aggctgactt gcggttgatc
     2281 tatctcgcgc tggcgcatat gatcaaattt cggggacact tcctcatcga gggggacctg
     2341 aacccagaca acagcgatgt cgacaaactc tttatccaac tggttcagac ttacaatcag
     2401 cttttcgaag agaacccgat caacgcatcc ggagttgacg ccaaagcaat cctgagcgct
     2461 aggctgtcca aatcccggcg gctcgaaaac ctcatcgcac agctccctgg ggagaagaag
     2521 aacggcctgt ttggtaatct tatcgccctg tcactcgggc tgacccccaa ctttaaatct
     2581 aacttcgacc tggccgaaga tgccaagctt caactgagca aagacaccta cgatgatgat
     2641 ctcgacaatc tgctggccca gatcggcgac cagtacgcag accttttttt ggcggcaaag
     2701 aacctgtcag acgccattct gctgagtgat attctgcgag tgaacacgga gatcaccaaa
     2761 gctccgctga gcgctagtat gatcaagcgc tatgatgagc accaccaaga cttgactttg
     2821 ctgaaggccc ttgtcagaca gcaactgcct gagaagtaca aggaaatttt cttcgatcag
     2881 tctaaaaatg gctacgccgg atacattgac ggcggagcaa gccaggagga attttacaaa
     2941 tttattaagc ccatcttgga aaaaatggac ggcaccgagg agctgctggt aaagcttaac
     3001 agagaagatc tgttgcgcaa acagcgcact ttcgacaatg gaagcatccc ccaccagatt
     3061 cacctgggcg aactgcacgc tatcctcagg cggcaagagg atttctaccc ctttttgaaa
     3121 gataacaggg aaaagattga gaaaatcctc acatttcgga taccctacta tgtaggcccc
     3181 ctcgcccggg gaaattccag attcgcgtgg atgactcgca aatcagaaga gaccatcact
     3241 ccctggaact tcgaggaagt cgtggataag ggggcctctg cccagtcctt catcgaaagg
     3301 atgactaact ttgataaaaa tctgcctaac gaaaaggtgc ttcctaaaca ctctctgctg
     3361 tacgagtact tcacagttta taacgagctc accaaggtca aatacgtcac agaagggatg
     3421 agaaagccag cattcctgtc tggagagcag aagaaagcta tcgtggacct cctcttcaag
     3481 acgaaccgga aagttaccgt gaaacagctc aaagaagact atttcaaaaa gattgaatgt
     3541 ttcgactctg ttgaaatcag cggagtggag gatcgcttca acgcatccct gggaacgtat
     3601 cacgatctcc tgaaaatcat taaagacaag gacttcctgg acaatgagga gaacgaggac
     3661 attcttgagg acattgtcct cacccttacg ttgtttgaag atagggagat gattgaagaa
     3721 cgcttgaaaa cttacgctca tctcttcgac gacaaagtca tgaaacagct caagaggcgc
     3781 cgatatacag gatgggggcg gctgtcaaga aaactgatca atgggatccg agacaagcag
     3841 agtggaaaga caatcctgga ttttcttaag tccgatggat ttgccaacag gaacttcatg
     3901 cagttgatcc atgatgactc tctcaccttt aaggaggaca tccagaaagc acaagtttct
     3961 ggccaggggg acagtcttca cgagcacatc gctaatcttg caggtagccc agctatcaaa
     4021 aagggaatac tgcagaccgt taaggtcgtg gatgaactcg tcaaagtaat gggaaggcat
     4081 aagcccgaga atatcgttat cgagatggcc cgagagaacc aaactaccca gaagggacag
     4141 aagaacagta gggaaaggat gaagaggatt gaagagggta taaaagaact ggggtcccaa
     4201 atccttaagg aacacccagt tgaaaacacc cagcttcaga atgagaagct ctacctgtac
     4261 tacctgcaga acggcaggga catgtacgtg gatcaggaac tggacatcaa tcggctctcc
     4321 gactacgacg tggatcatat cgtgccccag tcttttctca aagatgattc tattgataat
     4381 aaagtgttga caagatccga taaaaataga gggaagagtg ataacgtccc ctcagaagaa
     4441 gttgtcaaga aaatgaaaaa ttattggcgg cagctgctga acgccaaact gatcacacaa
     4501 cggaagttcg ataatctgac taaggctgaa cgaggtggcc tgtctgagtt ggataaagcc
     4561 ggcttcatca aaaggcagct tgttgagaca cgccagatca ccaagcacgt ggcccaaatt
     4621 ctcgattcac gcatgaacac caagtacgat gaaaatgaca aactgattcg agaggtgaaa
     4681 gttattactc tgaagtctaa gctggtctca gatttcagaa aggactttca gttttataag
     4741 gtgagagaga tcaacaatta ccaccatgcg catgatgcct acctgaatgc agtggtaggc
     4801 actgcactta tcaaaaaata tcccaagctt gaatctgaat ttgtttacgg agactataaa
     4861 gtgtacgatg ttaggaaaat gatcgcaaag tctgagcagg aaataggcaa ggccaccgct
     4921 aagtacttct tttacagcaa tattatgaat tttttcaaga ccgagattac actggccaat
     4981 ggagagattc ggaagcgacc acttatcgaa acaaacggag aaacaggaga aatcgtgtgg
     5041 gacaagggta gggatttcgc gacagtccgg aaggtcctgt ccatgccgca ggtgaacatc
     5101 gttaaaaaga ccgaagtaca gaccggaggc ttctccaagg aaagtatcct cccgaaaagg
     5161 aacagcgaca agctgatcgc acgcaaaaaa gattgggacc ccaagaaata cggcggattc
     5221 gattctccta cagtcgctta cagtgtactg gttgtggcca aagtggagaa agggaagtct
     5281 aaaaaactca aaagcgtcaa ggaactgctg ggcatcacaa tcatggagcg atcaagcttc
     5341 gaaaaaaacc ccatcgactt tctcgaggcg aaaggatata aagaggtcaa aaaagacctc
     5401 atcattaagc ttcccaagta ctctctcttt gagcttgaaa acggccggaa acgaatgctc
     5461 gctagtgcgg gcgagctgca gaaaggtaac gagctggcac tgccctctaa atacgttaat
     5521 ttcttgtatc tggccagcca ctatgaaaag ctcaaagggt ctcccgaaga taatgagcag
     5581 aagcagctgt tcgtggaaca acacaaacac taccttgatg agatcatcga gcaaataagc
     5641 gaattctcca aaagagtgat cctcgccgac gctaacctcg ataaggtgct ttctgcttac
     5701 aataagcaca gggataagcc catcagggag caggcagaaa acattatcca cttgtttact
     5761 ctgaccaact tgggcgcgcc tgcagccttc aagtacttcg acaccaccat agacagaaag
     5821 cggtacacct ctacaaagga ggtcctggac gccacactga ttcatcagtc aattacgggg
     5881 ctctatgaaa caagaatcga cctctctcag ctcggtggag acagcagggc tgaccccaag
     5941 aagaagagga aggtgaccgg tggtggaggc ggaggttctg ggggaggagg tagtggcggt
     6001 ggtggttcag gaggcggcgg aagcttggat ccaggtggag gtggaagcgg tgttaacatg
     6061 ggaaaatcaa aagaaatcag ccaagacctc agaaaaagaa ttgtagacct ccacaagtct
     6121 ggttcatcct tgggagcaat ttccaaacgc ctggcggtac cacgttcatc tgtgcaaaca
     6181 atagtacgca agtataaaca ccatgggacc acgcagccgt cataccgctg agcggccgct
     6241 atatgtacaa gtaactgcag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     6301 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     6361 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     6421 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     6481 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     6541 gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     6601 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6661 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     6721 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     6781 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6841 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6901 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6961 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     7021 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     7081 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     7141 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     7201 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     7261 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     7321 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     7381 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     7441 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     7501 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     7561 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     7621 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     7681 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     7741 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     7801 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     7861 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7921 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7981 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     8041 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     8101 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     8161 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     8221 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     8281 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     8341 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     8401 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     8461 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     8521 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     8581 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     8641 gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa
     8701 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     8761 aagctacaac aaggcaaggc ttgaccgaca attgatagcg gcatgctcgc gtt
//
