LOCUS       sequence-182081-        9387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Purification of Cas9 protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9387)
  AUTHORS   Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, Liu J, Zhang H, Liu 
            C, Ran Y, Gao C
  TITLE     Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 
            ribonucleoprotein complexes.
  JOURNAL   Nat Commun. 2017 Jan 18;8:14261. doi: 10.1038/ncomms14261.
  PUBMED    28098143
REFERENCE   2  (bases 1 to 9387)
  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: "Nat 
            Commun."; date: "2017-01-18"; volume: "8:14261. doi"; pages: " 
            10.1038/ncomms14261"
COMMENT     Sequence Label: pET28a-Cas9-His
FEATURES             Location/Qualifiers
     source          1..9387
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(37..84)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     80..98
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(151..168)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(175..195)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(196..4299)
                     /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             complement(196..4299)
                     /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             complement(4300..4320)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     RBS             4339..4361
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4376..4400
                     /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)."
     primer_bind     complement(4400..4419)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4401..4419)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4503..4522)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4728..4805
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4806..5888
                     /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     complement(4825..4844)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6697..6888
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6882..6904)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6990..7129
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     7045..7064
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(7144..7161)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7315..7903)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7395..7414)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(8014..8033)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             8025..8840
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             8025..8840
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(8098..8117)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(8932..9387)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9069..9090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     9281..9300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cccattcgcc aatccggata tagttcctcc tttcagcaaa aaacccctca agacccgttt
       61 agaggcccca aggggttatg ctagttattg ctcagcggtg gcagcagcca actcagcttc
      121 ctttcgggct ttgttagcag ccggatctca gtggtggtgg tggtggtgct cgagcacctt
      181 cctcttcttc ttggggtcac ctcctagctg actcaaatca atgcgtgttt cataaagacc
      241 agtgatggat tgatggataa gagtggcatc taaaacttct tttgtagacg tatatcgttt
      301 acgatcaatt gttgtatcaa aatatttaaa agcagcggga gctccaagat tcgtcaacgt
      361 aaataaatga ataatatttt ctgcttgttc acgtattggt ttgtctctat gtttgttata
      421 tgcactaaga actttatcta aattggcatc tgctaaaata acacgcttag aaaattcact
      481 gatttgctca ataatctcat ctaaataatg cttatgctgc tccacaaaca attgtttttg
      541 ttcgttatct tctggactac ccttcaactt ttcataatga ctagctaaat ataaaaaatt
      601 cacatatttg cttggcagag ccagctcatt tcctttttgt aattctccgg cactagccag
      661 catccgttta cgaccgtttt ctaactcaaa aagactatat ttaggtagtt taatgattaa
      721 gtctttttta acttccttat atcctttagc ttctaaaaag tcaatcggat ttttttcaaa
      781 ggaacttctt tccataattg tgatccctag taactcttta acggatttta acttcttcga
      841 tttccctttt tccaccttag caaccactag gactgaataa gctaccgttg gactatcaaa
      901 accaccatat ttttttggat cccagtcttt tttacgagca ataagcttgt ccgaatttct
      961 ttttggtaaa attgactcct tggagaatcc gcctgtctgt acttctgttt tcttgacaat
     1021 attgacttgg ggcatggaca atactttgcg cactgtggca aaatctcgcc ctttatccca
     1081 gacaatttct ccagtttccc cattagtttc gattagaggg cgtttgcgaa tctctccatt
     1141 tgcaagtgta atttctgttt tgaagaagtt catgatatta gagtaaaaga aatattttgc
     1201 ggttgctttg cctatttctt gctcagactt agcaatcatt ttacgaacat cataaacttt
     1261 ataatcacca tagacaaact ccgattcaag ttttggatat ttcttaatca aagcagttcc
     1321 aacgacggca tttagatacg catcatgggc atgatggtaa ttgttaatct cacgtacttt
     1381 atagaattgg aaatcttttc ggaagtcaga aactaattta gattttaagg taatcacttt
     1441 aacctctcga ataagtttat cattttcatc gtatttagta ttcatgcgac tatccaaaat
     1501 ttgtgccaca tgcttagtga tttggcgagt ttcaaccaat tggcgtttga taaaaccagc
     1561 tttatcaagt tcactcaaac ctccacgttc agctttcgtt aaattatcaa acttacgttg
     1621 agtgattaac ttggcgttta gaagttgtct ccaatagttt ttcatctttt tgactacttc
     1681 ttcacttgga acgttatccg atttaccacg atttttatca gaacgcgtta agaccttatt
     1741 gtctattgaa tcgtctttaa ggaaactttg tggaacaatg tgatcgacat cataatcact
     1801 taaacgatta atatctaatt cttggtccac atacatgtct cttccatttt ggagataata
     1861 gagatagagc ttttcatttt gcaattgagt attttcaaca ggatgctctt taagaatctg
     1921 acttcctaat tctttgatac cttcttcgat tcgtttcata cgctctcgcg aatttttctg
     1981 gcccttttga gttgtctgat tttcacgtgc catttcaata acgatatttt ctggcttatg
     2041 ccgccccatt actttgacca attcatcaac aacttttaca gtctgtaaaa tacctttttt
     2101 aatagcaggg ctaccagcta aatttgcaat atgttcatgt aaactatcgc cttgtccaga
     2161 cacttgtgct ttttgaatgt cttctttaaa tgtcaaacta tcatcatgga tcagctgcat
     2221 aaaattgcga ttggcaaaac catctgattt caaaaaatct aatattgttt tgccagattg
     2281 cttatcccta ataccattaa tcaattttcg agacaaacgt ccccaaccag tataacggcg
     2341 acgtttaagc tgtttcatca ccttatcatc aaagaggtga gcatatgttt taagtctttc
     2401 ctcaatcatc tccctatctt caaataaggt caatgttaaa acaatatcct ctaagatatc
     2461 ttcattttct tcattatcca aaaaatcttt atctttaata atttttagca aatcatggta
     2521 ggtacctaat gaagcattaa atctatcttc aactcctgaa atttcaacac tatcaaaaca
     2581 ttctattttt ttgaaataat cttcttttaa ttgcttaacg gttacttttc gatttgtttt
     2641 gaagagtaaa tcaacaatgg ctttcttctg ttcacctgaa agaaatgctg gttttcgcat
     2701 tccttcagta acatatttga cctttgtcaa ttcgttataa accgtaaaat actcataaag
     2761 caaactatgt tttggtagta ctttttcatt tggaagattt ttatcaaagt ttgtcatgcg
     2821 ttcaataaat gattgagctg aagcaccttt atcgacaact tcttcaaaat tccatggggt
     2881 aattgtttct tcagacttcc gagtcatcca tgcaaaacga ctattgccac gcgccaatgg
     2941 accaacataa taaggaattc gaaaagtcaa gattttttca atcttctcac gattgtcttt
     3001 taaaaatgga taaaagtctt cttgtcttct caaaatagca tgcagctcac ccaagtgaat
     3061 ttgatgggga atagagccgt tgtcaaaggt ccgttgcttg cgcagcaaat cttcacgatt
     3121 tagtttcacc aataattcct cagtaccatc cattttttct aaaattggtt tgataaattt
     3181 ataaaattct tcttggctag ctcccccatc aatataacct gcatatccgt tttttgattg
     3241 atcaaaaaag atttctttat acttttctgg aagttgttgt cgaactaaag cttttaaaag
     3301 agtcaagtct tgatgatgtt catcgtagcg tttaatcatt gaagctgata ggggagcctt
     3361 agttatttca gtatttactc ttaggatatc tgaaagtaaa atagcatctg ataaattctt
     3421 agctgccaaa aacaaatcag catattgatc tccaatttgc gccaataaat tatctaaatc
     3481 atcatcgtaa gtatcttttg aaagctgtaa tttagcatct tctgccaaat caaaatttga
     3541 tttaaaatta ggggtcaaac ccaatgacaa agcaatgaga ttcccaaata agccattttt
     3601 cttctcaccg gggagctgag caatgagatt ttctaatcgt cttgatttac tcaatcgtgc
     3661 agaaagaatc gctttagcat ctactccact tgcgttaata gggttttctt caaataattg
     3721 attgtaggtt tgtaccaact ggataaatag tttgtccaca tcactattat caggatttaa
     3781 atctccctca atcaaaaaat gaccacgaaa cttaatcata tgcgctaagg ccaaatagat
     3841 taagcgcaaa tccgctttat cagtagaatc taccaatttt tttcgcagat gatagatagt
     3901 tggatatttc tcatgataag caacttcatc tactatattt ccaaaaatag gatgacgttc
     3961 atgcttcttg tcttcttcca ccaaaaaaga ctcttcaagt cgatgaaaga aactatcatc
     4021 tactttcgcc atctcatttg aaaaaatctc ctgtagataa caaatacgat tcttccgacg
     4081 tgtatacctt ctacgagctg tccgtttgag acgagtcgct tccgctgtct ctccactgtc
     4141 aaataaaaga gcccctataa gatttttttt gatactgtgg cggtctgtat ttcccagaac
     4201 cttgaacttt ttagacggaa ccttatattc atcagtgatc accgcccatc cgacgctatt
     4261 tgtgccgata tctaagccta ttgagtattt cttatccatc accttcctct tcttcttggg
     4321 cataaacccc atggtatatc tccttcttaa agttaaacaa aattatttct agaggggaat
     4381 tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc
     4441 gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
     4501 cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
     4561 cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat
     4621 ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg
     4681 ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat
     4741 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     4801 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     4861 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     4921 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     4981 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     5041 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     5101 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     5161 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     5221 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     5281 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     5341 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     5401 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     5461 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     5521 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     5581 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     5641 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     5701 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     5761 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     5821 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     5881 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga
     5941 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     6001 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     6061 gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg
     6121 tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc
     6181 accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggcccc acgggtgcgc
     6241 atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag
     6301 cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg
     6361 acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg
     6421 aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc
     6481 tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc
     6541 tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca
     6601 tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc
     6661 cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc
     6721 cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag
     6781 ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt
     6841 taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     6901 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     6961 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     7021 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccatt
     7081 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
     7141 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     7201 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     7261 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     7321 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     7381 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     7441 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     7501 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     7561 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     7621 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     7681 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     7741 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     7801 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     7861 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     7921 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa
     7981 caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac
     8041 gggaaacgtc ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     8101 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga
     8161 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     8221 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     8281 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag
     8341 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     8401 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     8461 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     8521 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt
     8581 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     8641 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     8701 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     8761 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     8821 tgctcgatga gtttttctaa gaattaattc atgagcggat acatatttga atgtatttag
     8881 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     8941 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     9001 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     9061 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     9121 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     9181 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     9241 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     9301 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     9361 cgcttaatgc gccgctacag ggcgcgt
//
