LOCUS       sequence-486082-       10136 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Bacterial expression of SpyCas9 (WT) with 2xSV40 nuclear 
            localization sequence for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10136)
  AUTHORS   Shi H, Al-Sayyad N, Wasko KM, Trinidad MI, Doherty EE, Vohra K, 
            Boger RS, Colognori D, Cofsky JC, Skopintsev P, Bryant Z, Doudna JA
  TITLE     Rapid two-step target capture ensures efficient CRISPR-Cas9-guided 
            genome editing.
  JOURNAL   Mol Cell. 2025 May 1;85(9):1730-1742.e9. doi: 
            10.1016/j.molcel.2025.03.024. Epub 2025 Apr 23.
  PUBMED    40273916
REFERENCE   2  (bases 1 to 10136)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2025.03.024"; journalName: "Mol Cell"; date: 
            "2025-05-1- 1"; volume: "85"; issue: "9"; pages: "1730-1742.e9"
COMMENT     Sequence Label: pHS71_10xHis_MBP_TEV_WT-Cas9_2xNLS
FEATURES             Location/Qualifiers
     source          1..10136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     349..368
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     452..471
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        452..470
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             559..581
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             601..630
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             640..1740
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1714..1737
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1795..1815
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1822..5925
                     /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             1822..5925
                     /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             5932..5952
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5959..5979
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6117..6135)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6131..6178
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6544..6549,6550..6566,6567..6572))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6599..6617)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6685..6789
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6790..6858,6859..7650)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7008..7027)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7821..8409
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8310..8329
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8563..8580
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8595..8734
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8660..8679)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8820..8842
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8836..9027)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
       61 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
      121 gactgggttg aaggctctca agggcatcgg tcgacgctct cccttatgcg actcctgcat
      181 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
      241 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
      301 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
      361 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
      421 gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg gagaccacaa
      481 cggtttccct ctagtgccgg ctccggagag ctctttaatt aagcggccgc cctgcaggac
      541 tcgagttcta gaaataattt tgtttaactt taagaaggag atatacatat gaaatcttct
      601 caccatcacc atcaccatca ccatcaccat ggttcttcta tgaaaatcga agaaggtaaa
      661 ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt cggtaagaaa
      721 ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa
      781 ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc
      841 tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag
      901 gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac
      961 ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca
     1021 aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg
     1081 ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt
     1141 tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct
     1201 ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca
     1261 gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc
     1321 aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta
     1381 ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt
     1441 aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact
     1501 gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct
     1561 tacgaggaag agttggcgaa agatccacgt attgccgcca ctatggaaaa cgcccagaaa
     1621 ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg
     1681 gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact
     1741 aattcgagct cgaacaacaa caacaataac aataacaaca acctcgggat cgaggaaaac
     1801 ctgtacttcc aatccaatgc aatggataag aaatactcaa taggcttaga tatcggcaca
     1861 aatagcgtcg gatgggcggt gatcactgat gaatataagg ttccgtctaa aaagttcaag
     1921 gttctgggaa atacagaccg ccacagtatc aaaaaaaatc ttataggggc tcttttattt
     1981 gacagtggag agacagcgga agcgactcgt ctcaaacgga cagctcgtag aaggtataca
     2041 cgtcggaaga atcgtatttg ttatctacag gagatttttt caaatgagat ggcgaaagta
     2101 gatgatagtt tctttcatcg acttgaagag tcttttttgg tggaagaaga caagaagcat
     2161 gaacgtcatc ctatttttgg aaatatagta gatgaagttg cttatcatga gaaatatcca
     2221 actatctatc atctgcgaaa aaaattggta gattctactg ataaagcgga tttgcgctta
     2281 atctatttgg ccttagcgca tatgattaag tttcgtggtc attttttgat tgagggagat
     2341 ttaaatcctg ataatagtga tgtggacaaa ctatttatcc agttggtaca aacctacaat
     2401 caattatttg aagaaaaccc tattaacgca agtggagtag atgctaaagc gattctttct
     2461 gcacgattga gtaaatcaag acgattagaa aatctcattg ctcagctccc cggtgagaag
     2521 aaaaatggct tatttgggaa tctcattgct ttgtcattgg gtttgacccc taattttaaa
     2581 tcaaattttg atttggcaga agatgctaaa ttacagcttt caaaagatac ttacgatgat
     2641 gatttagata atttattggc gcaaattgga gatcaatatg ctgatttgtt tttggcagct
     2701 aagaatttat cagatgctat tttactttca gatatcctaa gagtaaatac tgaaataact
     2761 aaggctcccc tatcagcttc aatgattaaa cgctacgatg aacatcatca agacttgact
     2821 cttttaaaag ctttagttcg acaacaactt ccagaaaagt ataaagaaat cttttttgat
     2881 caatcaaaaa acggatatgc aggttatatt gatgggggag ctagccaaga agaattttat
     2941 aaatttatca aaccaatttt agaaaaaatg gatggtactg aggaattatt ggtgaaacta
     3001 aatcgtgaag atttgctgcg caagcaacgg acctttgaca acggctctat tccccatcaa
     3061 attcacttgg gtgagctgca tgctattttg agaagacaag aagactttta tccattttta
     3121 aaagacaatc gtgagaagat tgaaaaaatc ttgacttttc gaattcctta ttatgttggt
     3181 ccattggcgc gtggcaatag tcgttttgca tggatgactc ggaagtctga agaaacaatt
     3241 accccatgga attttgaaga agttgtcgat aaaggtgctt cagctcaatc atttattgaa
     3301 cgcatgacaa actttgataa aaatcttcca aatgaaaaag tactaccaaa acatagtttg
     3361 ctttatgagt attttacggt ttataacgaa ttgacaaagg tcaaatatgt tactgaagga
     3421 atgcgaaaac cagcatttct ttcaggtgaa cagaagaaag ccattgttga tttactcttc
     3481 aaaacaaatc gaaaagtaac cgttaagcaa ttaaaagaag attatttcaa aaaaatagaa
     3541 tgttttgata gtgttgaaat ttcaggagtt gaagatagat ttaatgcttc attaggtacc
     3601 taccatgatt tgctaaaaat tattaaagat aaagattttt tggataatga agaaaatgaa
     3661 gatatcttag aggatattgt tttaacattg accttatttg aagataggga gatgattgag
     3721 gaaagactta aaacatatgc tcacctcttt gatgataagg tgatgaaaca gcttaaacgt
     3781 cgccgttata ctggttgggg acgtttgtct cgaaaattga ttaatggtat tagggataag
     3841 caatctggca aaacaatatt agattttttg aaatcagatg gttttgccaa tcgcaatttt
     3901 atgcagctga tccatgatga tagtttgaca tttaaagaag acattcaaaa agcacaagtg
     3961 tctggacaag gcgatagttt acatgaacat attgcaaatt tagctggtag ccctgctatt
     4021 aaaaaaggta ttttacagac tgtaaaagtt gttgatgaat tggtcaaagt aatggggcgg
     4081 cataagccag aaaatatcgt tattgaaatg gcacgtgaaa atcagacaac tcaaaagggc
     4141 cagaaaaatt cgcgagagcg tatgaaacga atcgaagaag gtatcaaaga attaggaagt
     4201 cagattctta aagagcatcc tgttgaaaat actcaattgc aaaatgaaaa gctctatctc
     4261 tattatctcc aaaatggaag agacatgtat gtggaccaag aattagatat taatcgttta
     4321 agtgattatg atgtcgatca cattgttcca caaagtttcc ttaaagacga ttcaatagac
     4381 aataaggtct taacgcgttc tgataaaaat cgtggtaaat cggataacgt tccaagtgaa
     4441 gaagtagtca aaaagatgaa aaactattgg agacaacttc taaacgccaa gttaatcact
     4501 caacgtaagt ttgataattt aacgaaagct gaacgtggag gtttgagtga acttgataaa
     4561 gctggtttta tcaaacgcca attggttgaa actcgccaaa tcactaagca tgtggcacaa
     4621 attttggata gtcgcatgaa tactaaatac gatgaaaatg ataaacttat tcgagaggtt
     4681 aaagtgatta ccttaaaatc taaattagtt tctgacttcc gaaaagattt ccaattctat
     4741 aaagtacgtg agattaacaa ttaccatcat gcccatgatg cgtatctaaa tgccgtcgtt
     4801 ggaactgctt tgattaagaa atatccaaaa cttgaatcgg agtttgtcta tggtgattat
     4861 aaagtttatg atgttcgtaa aatgattgct aagtctgagc aagaaatagg caaagcaacc
     4921 gcaaaatatt tcttttactc taatatcatg aacttcttca aaacagaaat tacacttgca
     4981 aatggagaga ttcgcaaacg ccctctaatc gaaactaatg gggaaactgg agaaattgtc
     5041 tgggataaag ggcgagattt tgccacagtg cgcaaagtat tgtccatgcc ccaagtcaat
     5101 attgtcaaga aaacagaagt acagacaggc ggattctcca aggagtcaat tttaccaaaa
     5161 agaaattcgg acaagcttat tgctcgtaaa aaagactggg atccaaaaaa atatggtggt
     5221 tttgatagtc caacggtagc ttattcagtc ctagtggttg ctaaggtgga aaaagggaaa
     5281 tcgaagaagt taaaatccgt taaagagtta ctagggatca caattatgga aagaagttcc
     5341 tttgaaaaaa atccgattga ctttttagaa gctaaaggat ataaggaagt taaaaaagac
     5401 ttaatcatta aactacctaa atatagtctt tttgagttag aaaacggtcg taaacggatg
     5461 ctggctagtg ccggagaatt acaaaaagga aatgagctgg ctctgccaag caaatatgtg
     5521 aattttttat atttagctag tcattatgaa aagttgaagg gtagtccaga agataacgaa
     5581 caaaaacaat tgtttgtgga gcagcataag cattatttag atgagattat tgagcaaatc
     5641 agtgaatttt ctaagcgtgt tattttagca gatgccaatt tagataaagt tcttagtgca
     5701 tataacaaac atagagacaa accaatacgt gaacaagcag aaaatattat tcatttattt
     5761 acgttgacga atcttggagc tcccgctgct tttaaatatt ttgatacaac aattgatcgt
     5821 aaacgatata cgtctacaaa agaagtttta gatgccactc ttatccatca atccatcact
     5881 ggtctttatg aaacacgcat tgatttgagt cagctaggag gtgacgggtc acctaagaaa
     5941 aaacgaaaag ttgaggatcc taaaaagaaa cgaaaagttg ataccggtta ataacattgg
     6001 aagtggataa cggatccgcg atcgcggcgc gccacctggt ggccggccgg taccacgcgt
     6061 gcgcgctgat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     6121 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     6181 gaaaggagga actatatccg gatatccaca ggacgggtgt ggtcgccatg atcgcgtagt
     6241 cgatagtggc tccaagtagc gaagcgagca ggactgggcg gcggccaaag cggtcggaca
     6301 gtgctccgag aacgggtgcg catagaaatt gcatcaacgc atatagcgct agcagcacgc
     6361 catagtgact ggcgatgctg tcggaatgga cgatatcccg caagaggccc ggcagtaccg
     6421 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg
     6481 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
     6541 cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc
     6601 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     6661 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     6721 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata acattgaaaa
     6781 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     6841 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     6901 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     6961 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     7021 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     7081 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     7141 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     7201 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     7261 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     7321 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     7381 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     7441 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     7501 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     7561 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     7621 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     7681 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     7741 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     7801 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     7861 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     7921 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     7981 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     8041 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     8101 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     8161 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     8221 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     8281 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     8341 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     8401 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     8461 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     8521 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     8581 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     8641 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     8701 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     8761 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     8821 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     8881 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     8941 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     9001 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     9061 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     9121 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     9181 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     9241 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     9301 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     9361 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     9421 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     9481 catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga
     9541 gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg
     9601 caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct
     9661 tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca
     9721 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca
     9781 taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc
     9841 gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac
     9901 gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct
     9961 cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg
    10021 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
    10081 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaa
//
