LOCUS       sequence-273580-        8727 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Himar-dCas9 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8727)
  AUTHORS   Chen SP, Wang HH
  TITLE     An Engineered Cas-Transposon System for Programmable and 
            Site-Directed DNA Transpositions.
  JOURNAL   CRISPR J. 2019 Dec;2(6):376-394. doi: 10.1089/crispr.2019.0030. Epub
            2019 Nov 19.
  PUBMED    31742433
REFERENCE   2  (bases 1 to 8727)
  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: 
            "10.1089/crispr.2019.0030"; journalName: "CRISPR J"; date: 
            "2019-12"; volume: "2"; issue: "6"; pages: "376-394"
COMMENT     Sequence Label: pHdCas9-mammalian
FEATURES             Location/Qualifiers
     source          1..8727
                     /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"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      403..412
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             412..477
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             484..504
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1633..1656,1657..1659,1660..4146,4147..4149,
                     4150..5736)
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(1633..5031,5032..5034,5035..5631,5632,5633..5635,
                     5636..5637,5638..5640,5641..5733)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5742..5759
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5782..5799)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5788..6012
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6083..6099)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6083..6099)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6096..6118)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6107..6123
                     /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(6131..6137,6138..6155,6156..6161))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6176..6197
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6314..6331)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6485..7073)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6565..6584)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7244..8035,8036..8104))
                     /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     7867..7886
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8105..8209)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8288..8307)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8479..8727,1..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 ggccgctaat acgactcact atagggagag ccgccaccat ggactacaaa
      421 gaccatgacg gtgattataa agatcatgac atcgattaca aggatgacga tgacaagatg
      481 gcccccaaga agaagaggaa ggtgggcatt caccgcgggg tacctggagg ttctggatcc
      541 atggaaaaaa aggaatttcg tgttttgata aaatactgtt ttctgaaggg aaaaaataca
      601 gtggaagcaa aaacttggct tgataatgag tttccggact ctgccccagg gaaatcaaca
      661 ataattgatt ggtatgcaaa attcaagcgt ggtgaaatga gcacggagga cggtgaacgc
      721 agtggacgcc cgaaagaggt ggttaccgac gaaaacatca aaaaaatcca caaaatgatt
      781 ttgaatgacc gtaaaatgaa gttgatcgag atagcagagg ccttaaagat atcaaaggaa
      841 cgtgttggtc atatcattca tcaatatttg gatatgcgga agctctgtgc gaaatgggtg
      901 ccgcgcgagc tcacatttga ccaaaaacaa cgacgtgttg atgattctaa gcggtgtttg
      961 cagctgttaa ctcgtaatac acccgagttt ttccgtcgat atgtgacaat ggatgaaaca
     1021 tggctccatc actacactcc tgagtccaat cgacagtcgg ctgagtggac agcgaccggt
     1081 gaaccgtctc cgaagcgtgg aaagactcaa aagtccgctg gcaaagtaat ggcctctgtt
     1141 ttttgggatg cgcatggaat aatttttatc gattatcttg agaagggaaa aaccatcaac
     1201 agtgactatt atatggcgtt attggagcgt ttgaaggtcg aaatcgcggc aaaacggccc
     1261 cacatgaaga agaaaaaagt gttgttccac caagacaacg caccgtgcca caagtcattg
     1321 agaacgatgg caaaaattca tgaattgggc ttcgaattgc ttccccaccc gccgtattct
     1381 ccagatctgg cccccagcga ctttttcttg ttctcagacc tcaaaaggat gctcgcaggg
     1441 aaaaaatttg gctgcaatga agaggtgatc gccgaaactg aggcctattt tgaggcaaaa
     1501 ccgaaggagt actaccaaaa tggtatcaaa aaattggaag gtcgttataa tcgttgtatc
     1561 gctcttgaag ggaactatgt tgaaagcggc agcgagactc ccgggacctc agagtccgcc
     1621 acacccgaaa gtgataaaaa gtattctatt ggtttagcta tcggcactaa ttccgttgga
     1681 tgggctgtca taaccgatga atacaaagta ccttcaaaga aatttaaggt gttggggaac
     1741 acagaccgtc attcgattaa aaagaatctt atcggtgccc tcctattcga tagtggcgaa
     1801 acggcagagg cgactcgcct gaaacgaacc gctcggagaa ggtatacacg tcgcaagaac
     1861 cgaatatgtt acttacaaga aatttttagc aatgagatgg ccaaagttga cgattctttc
     1921 tttcaccgtt tggaagagtc cttccttgtc gaagaggaca agaaacatga acggcacccc
     1981 atctttggaa acatagtaga tgaggtggca tatcatgaaa agtacccaac gatttatcac
     2041 ctcagaaaaa agctagttga ctcaactgat aaagcggacc tgaggttaat ctacttggct
     2101 cttgcccata tgataaagtt ccgtgggcac tttctcattg agggtgatct aaatccggac
     2161 aactcggatg tcgacaaact gttcatccag ttagtacaaa cctataatca gttgtttgaa
     2221 gagaacccta taaatgcaag tggcgtggat gcgaaggcta ttcttagcgc ccgcctctct
     2281 aaatcccgac ggctagaaaa cctgatcgca caattacccg gagagaagaa aaatgggttg
     2341 ttcggtaacc ttatagcgct ctcactaggc ctgacaccaa attttaagtc gaacttcgac
     2401 ttagctgaag atgccaaatt gcagcttagt aaggacacgt acgatgacga tctcgacaat
     2461 ctactggcac aaattggaga tcagtatgcg gacttatttt tggctgccaa aaaccttagc
     2521 gatgcaatcc tcctatctga catactgaga gttaatactg agattaccaa ggcgccgtta
     2581 tccgcttcaa tgatcaaaag gtacgatgaa catcaccaag acttgacact tctcaaggcc
     2641 ctagtccgtc agcaactgcc tgagaaatat aaggaaatat tctttgatca gtcgaaaaac
     2701 gggtacgcag gttatattga cggcggagcg agtcaagagg aattctacaa gtttatcaaa
     2761 cccatattag agaagatgga tgggacggaa gagttgcttg taaaactcaa tcgcgaagat
     2821 ctactgcgaa agcagcggac tttcgacaac ggtagcattc cacatcaaat ccacttaggc
     2881 gaattgcatg ctatacttag aaggcaggag gatttttatc cgttcctcaa agacaatcgt
     2941 gaaaagattg agaaaatcct aacctttcgc ataccttact atgtgggacc cctggcccga
     3001 gggaactctc ggttcgcatg gatgacaaga aagtccgaag aaacgattac tccatggaat
     3061 tttgaggaag ttgtcgataa aggtgcgtca gctcaatcgt tcatcgagag gatgaccaac
     3121 tttgacaaga atttaccgaa cgaaaaagta ttgcctaagc acagtttact ttacgagtat
     3181 ttcacagtgt acaatgaact cacgaaagtt aagtatgtca ctgagggcat gcgtaaaccc
     3241 gcctttctaa gcggagaaca gaagaaagca atagtagatc tgttattcaa gaccaaccgc
     3301 aaagtgacag ttaagcaatt gaaagaggac tactttaaga aaattgaatg cttcgattct
     3361 gtcgagatct ccggggtaga agatcgattt aatgcgtcac ttggtacgta tcatgacctc
     3421 ctaaagataa ttaaagataa ggacttcctg gataacgaag agaatgaaga tatcttagaa
     3481 gatatagtgt tgactcttac cctctttgaa gatcgggaaa tgattgagga aagactaaaa
     3541 acatacgctc acctgttcga cgataaggtt atgaaacagt taaagaggcg tcgctatacg
     3601 ggctggggac gattgtcgcg gaaacttatc aacgggataa gagacaagca aagtggtaaa
     3661 actattctcg attttctaaa gagcgacggc ttcgccaata ggaactttat gcagctgatc
     3721 catgatgact ctttaacctt caaagaggat atacaaaagg cacaggtttc cggacaaggg
     3781 gactcattgc acgaacatat tgcgaatctt gctggttcgc cagccatcaa aaagggcata
     3841 ctccagacag tcaaagtagt ggatgagcta gttaaggtca tgggacgtca caaaccggaa
     3901 aacattgtaa tcgagatggc acgcgaaaat caaacgactc agaaggggca aaaaaacagt
     3961 cgagagcgga tgaagagaat agaagagggt attaaagaac tgggcagcca gatcttaaag
     4021 gagcatcctg tggaaaatac ccaattgcag aacgagaaac tttacctcta ttacctacaa
     4081 aatggaaggg acatgtatgt tgatcaggaa ctggacataa accgtttatc tgattacgac
     4141 gtcgatgcca ttgtacccca atcctttttg aaggacgatt caatcgacaa taaagtgctt
     4201 acacgctcgg ataagaaccg agggaaaagt gacaatgttc caagcgagga agtcgtaaag
     4261 aaaatgaaga actattggcg gcagctccta aatgcgaaac tgataacgca aagaaagttc
     4321 gataacttaa ctaaagctga gaggggtggc ttgtctgaac ttgacaaggc cggatttatt
     4381 aaacgtcagc tcgtggaaac ccgccaaatc acaaagcatg ttgcacagat actagattcc
     4441 cgaatgaata cgaaatacga cgagaacgat aagctgattc gggaagtcaa agtaatcact
     4501 ttaaagtcaa aattggtgtc ggacttcaga aaggattttc aattctataa agttagggag
     4561 ataaataact accaccatgc gcacgacgct tatcttaatg ccgtcgtagg gaccgcactc
     4621 attaagaaat acccgaagct agaaagtgag tttgtgtatg gtgattacaa agtttatgac
     4681 gtccgtaaga tgatcgcgaa aagcgaacag gagataggca aggctacagc caaatacttc
     4741 ttttattcta acattatgaa tttctttaag acggaaatca ctctggcaaa cggagagata
     4801 cgcaaacgac ctttaattga aaccaatggg gagacaggtg aaatcgtatg ggataagggc
     4861 cgggacttcg cgacggtgag aaaagttttg tccatgcccc aagtcaacat agtaaagaaa
     4921 actgaggtgc agaccggagg gttttcaaag gaatcgattc ttccaaaaag gaatagtgat
     4981 aagctcatcg ctcgtaaaaa ggactgggac ccgaaaaagt acggtggctt cgatagccct
     5041 acagttgcct attctgtcct agtagtggca aaagttgaga agggaaaatc caagaaactg
     5101 aagtcagtca aagaattatt ggggataacg attatggagc gctcgtcttt tgaaaagaac
     5161 cccatcgact tccttgaggc gaaaggttac aaggaagtaa aaaaggatct cataattaaa
     5221 ctaccaaagt atagtctgtt tgagttagaa aatggccgaa aacggatgtt ggctagcgcc
     5281 ggagagcttc aaaaggggaa cgaactcgca ctaccgtcta aatacgtgaa tttcctgtat
     5341 ttagcgtccc attacgagaa gttgaaaggt tcacctgaag ataacgaaca gaagcaactt
     5401 tttgttgagc agcacaaaca ttatctcgac gaaatcatag agcaaatttc ggaattcagt
     5461 aagagagtca tcctagctga tgccaatctg gacaaagtat taagcgcata caacaagcac
     5521 agggataaac ccatacgtga gcaggcggaa aatattatcc atttgtttac tcttaccaac
     5581 ctcggcgctc cagccgcatt caagtatttt gacacaacga tagatcgcaa acgatacact
     5641 tctaccaagg aggtgctaga cgcgacactg attcaccaat ccatcacggg attatatgaa
     5701 actcggatag atttgtcaca gcttgggggt gactgaccgg tcatcatcac catcaccatt
     5761 gagtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     5821 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     5881 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     5941 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     6001 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctcgataccg tcgacctcta
     6061 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     6121 caattccaca caacatacga gccggaagca taaagtgtaa agcctagggt gcctaatgag
     6181 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     6241 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6301 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     6361 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     6421 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     6481 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6541 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6601 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6661 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6721 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6781 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6841 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6901 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6961 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     7021 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     7081 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     7141 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     7201 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     7261 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     7321 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     7381 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     7441 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     7501 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     7561 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     7621 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     7681 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     7741 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     7801 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     7861 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     7921 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7981 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     8041 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     8101 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     8161 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     8221 gaaaagtgcc acctgacgtc gacggatcgg gagatcgatc tcccgatccc ctagggtcga
     8281 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     8341 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
     8401 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
     8461 gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     8521 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     8581 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     8641 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     8701 cccacttggc agtacatcaa gtgtatc
//
