LOCUS       sequence-211944-        9286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of NLS-Cas9-6xHis in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9286)
  AUTHORS   Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder
            ML, Joung JK, Chen ZY, Liu DR
  TITLE     Cationic lipid-mediated delivery of proteins enables efficient 
            protein-based genome editing in vitro and in vivo.
  JOURNAL   Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 
            2014 Oct 30.
  PUBMED    25357182
REFERENCE   2  (bases 1 to 9286)
  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: "10.1038/nbt.3081"; 
            journalName: "Nat Biotechnol"; date: "2015-01"; volume: "33"; issue:
            "1"; pages: "73-80"
COMMENT     Sequence Label: pET-NLS-Cas9-6xHis
FEATURES             Location/Qualifiers
     source          1..9286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     RBS             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             74..94
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             95..4198
                     /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             95..4198
                     /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             4199..4216
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4235..4279
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4317..4335)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4331..4378
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4415..4870
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4502..4521)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4712..4733
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4985..5776,5777..5845))
                     /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     5608..5627
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5846..5938)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6019..6607
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6508..6527
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6761..6778
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6793..6932
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6858..6877)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7018..7040
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7034..7225)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7797..8879)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     8841..8860
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8880..8957)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9163..9182
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(9270..9286,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(9270..9286,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgcccaaga agaagaggaa ggtgatggat aagaaatact caataggctt
      121 agatatcggc acaaatagcg tcggatgggc ggtgatcact gatgaatata aggttccgtc
      181 taaaaagttc aaggttctgg gaaatacaga ccgccacagt atcaaaaaaa atcttatagg
      241 ggctctttta tttgacagtg gagagacagc ggaagcgact cgtctcaaac ggacagctcg
      301 tagaaggtat acacgtcgga agaatcgtat ttgttatcta caggagattt tttcaaatga
      361 gatggcgaaa gtagatgata gtttctttca tcgacttgaa gagtcttttt tggtggaaga
      421 agacaagaag catgaacgtc atcctatttt tggaaatata gtagatgaag ttgcttatca
      481 tgagaaatat ccaactatct atcatctgcg aaaaaaattg gtagattcta ctgataaagc
      541 ggatttgcgc ttaatctatt tggccttagc gcatatgatt aagtttcgtg gtcatttttt
      601 gattgaggga gatttaaatc ctgataatag tgatgtggac aaactattta tccagttggt
      661 acaaacctac aatcaattat ttgaagaaaa ccctattaac gcaagtggag tagatgctaa
      721 agcgattctt tctgcacgat tgagtaaatc aagacgatta gaaaatctca ttgctcagct
      781 ccccggtgag aagaaaaatg gcttatttgg gaatctcatt gctttgtcat tgggtttgac
      841 ccctaatttt aaatcaaatt ttgatttggc agaagatgct aaattacagc tttcaaaaga
      901 tacttacgat gatgatttag ataatttatt ggcgcaaatt ggagatcaat atgctgattt
      961 gtttttggca gctaagaatt tatcagatgc tattttactt tcagatatcc taagagtaaa
     1021 tactgaaata actaaggctc ccctatcagc ttcaatgatt aaacgctacg atgaacatca
     1081 tcaagacttg actcttttaa aagctttagt tcgacaacaa cttccagaaa agtataaaga
     1141 aatctttttt gatcaatcaa aaaacggata tgcaggttat attgatgggg gagctagcca
     1201 agaagaattt tataaattta tcaaaccaat tttagaaaaa atggatggta ctgaggaatt
     1261 attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa cggacctttg acaacggctc
     1321 tattccccat caaattcact tgggtgagct gcatgctatt ttgagaagac aagaagactt
     1381 ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa atcttgactt ttcgaattcc
     1441 ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt gcatggatga ctcggaagtc
     1501 tgaagaaaca attaccccat ggaattttga agaagttgtc gataaaggtg cttcagctca
     1561 atcatttatt gaacgcatga caaactttga taaaaatctt ccaaatgaaa aagtactacc
     1621 aaaacatagt ttgctttatg agtattttac ggtttataac gaattgacaa aggtcaaata
     1681 tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt gaacagaaga aagccattgt
     1741 tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag caattaaaag aagattattt
     1801 caaaaaaata gaatgttttg atagtgttga aatttcagga gttgaagata gatttaatgc
     1861 ttcattaggt acctaccatg atttgctaaa aattattaaa gataaagatt ttttggataa
     1921 tgaagaaaat gaagatatct tagaggatat tgttttaaca ttgaccttat ttgaagatag
     1981 ggagatgatt gaggaaagac ttaaaacata tgctcacctc tttgatgata aggtgatgaa
     2041 acagcttaaa cgtcgccgtt atactggttg gggacgtttg tctcgaaaat tgattaatgg
     2101 tattagggat aagcaatctg gcaaaacaat attagatttt ttgaaatcag atggttttgc
     2161 caatcgcaat tttatgcagc tgatccatga tgatagtttg acatttaaag aagacattca
     2221 aaaagcacaa gtgtctggac aaggcgatag tttacatgaa catattgcaa atttagctgg
     2281 tagccctgct attaaaaaag gtattttaca gactgtaaaa gttgttgatg aattggtcaa
     2341 agtaatgggg cggcataagc cagaaaatat cgttattgaa atggcacgtg aaaatcagac
     2401 aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa cgaatcgaag aaggtatcaa
     2461 agaattagga agtcagattc ttaaagagca tcctgttgaa aatactcaat tgcaaaatga
     2521 aaagctctat ctctattatc tccaaaatgg aagagacatg tatgtggacc aagaattaga
     2581 tattaatcgt ttaagtgatt atgatgtcga tcacattgtt ccacaaagtt tccttaaaga
     2641 cgattcaata gacaataagg tcttaacgcg ttctgataaa aatcgtggta aatcggataa
     2701 cgttccaagt gaagaagtag tcaaaaagat gaaaaactat tggagacaac ttctaaacgc
     2761 caagttaatc actcaacgta agtttgataa tttaacgaaa gctgaacgtg gaggtttgag
     2821 tgaacttgat aaagctggtt ttatcaaacg ccaattggtt gaaactcgcc aaatcactaa
     2881 gcatgtggca caaattttgg atagtcgcat gaatactaaa tacgatgaaa atgataaact
     2941 tattcgagag gttaaagtga ttaccttaaa atctaaatta gtttctgact tccgaaaaga
     3001 tttccaattc tataaagtac gtgagattaa caattaccat catgcccatg atgcgtatct
     3061 aaatgccgtc gttggaactg ctttgattaa gaaatatcca aaacttgaat cggagtttgt
     3121 ctatggtgat tataaagttt atgatgttcg taaaatgatt gctaagtctg agcaagaaat
     3181 aggcaaagca accgcaaaat atttctttta ctctaatatc atgaacttct tcaaaacaga
     3241 aattacactt gcaaatggag agattcgcaa acgccctcta atcgaaacta atggggaaac
     3301 tggagaaatt gtctgggata aagggcgaga ttttgccaca gtgcgcaaag tattgtccat
     3361 gccccaagtc aatattgtca agaaaacaga agtacagaca ggcggattct ccaaggagtc
     3421 aattttacca aaaagaaatt cggacaagct tattgctcgt aaaaaagact gggatccaaa
     3481 aaaatatggt ggttttgata gtccaacggt agcttattca gtcctagtgg ttgctaaggt
     3541 ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag ttactaggga tcacaattat
     3601 ggaaagaagt tcctttgaaa aaaatccgat tgacttttta gaagctaaag gatataagga
     3661 agttaaaaaa gacttaatca ttaaactacc taaatatagt ctttttgagt tagaaaacgg
     3721 tcgtaaacgg atgctggcta gtgccggaga attacaaaaa ggaaatgagc tggctctgcc
     3781 aagcaaatat gtgaattttt tatatttagc tagtcattat gaaaagttga agggtagtcc
     3841 agaagataac gaacaaaaac aattgtttgt tgagcagcat aagcattatt tagatgagat
     3901 tattgagcaa atcagtgaat tttctaagcg tgttatttta gcagatgcca atttagataa
     3961 agttcttagt gcatataaca aacatagaga caaaccaata cgtgaacaag cagaaaatat
     4021 tattcattta tttacgttga cgaatcttgg agctcccgct gcttttaaat attttgatac
     4081 aacaattgat cgtaaacgat atacgtctac aaaagaagtt ttagatgcca ctcttatcca
     4141 tcaatccatc actggtcttt atgaaacacg cattgatttg agtcagctag gaggtgacca
     4201 tcaccaccac catcactaat gactcgagtc tggtaaagaa accgctgctg cgaaatttga
     4261 acgccagcac atggactcgt ctactagcgc agcttaatta acctaggctg ctgccaccgc
     4321 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     4381 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     4441 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     4501 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     4561 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4621 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4681 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4741 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4801 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4861 tttacaattt ctggcggcac gatggcatga gattatcaaa aaggatcttc acctagatcc
     4921 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4981 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5041 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5101 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5161 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5221 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5281 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5341 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5401 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5461 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5521 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5581 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5641 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5701 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5761 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5821 cgacacggaa atgttgaata ctcatactct tcctttttca atcatgattg aagcatttat
     5881 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5941 ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6001 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6061 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6121 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     6181 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6241 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6301 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6361 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6421 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6481 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6541 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6601 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     6661 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     6721 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     6781 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     6841 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     6901 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     6961 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     7021 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     7081 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     7141 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     7201 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     7261 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     7321 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     7381 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     7441 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     7501 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     7561 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     7621 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     7681 tgctagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     7741 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     7801 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     7861 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     7921 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     7981 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     8041 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     8101 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     8161 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     8221 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     8281 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     8341 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     8401 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     8461 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     8521 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     8581 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     8641 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     8701 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     8761 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     8821 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     8881 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     8941 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     9001 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     9061 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     9121 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     9181 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     9241 gatcgagatc gatctcgatc ccgcgaaatt aatacgactc actata
//
