LOCUS       sequence-177332-       10205 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of modified SpyCas9 in e.coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10205)
  AUTHORS   Staahl BT, Benekareddy M, Coulon-Bainier C, Banfal AA, Floor SN, 
            Sabo JK, Urnes C, Munares GA, Ghosh A, Doudna JA
  TITLE     Efficient genome editing in the mouse brain by local delivery of 
            engineered Cas9 ribonucleoprotein complexes.
  JOURNAL   Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3806.
  PUBMED    28191903
REFERENCE   2  (bases 1 to 10205)
  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 
            Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3806."
COMMENT     Sequence Label: 2xNLS-pMJ915v2
FEATURES             Location/Qualifiers
     source          1..10205
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     993..1012
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1096..1115
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1096..1114
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1203..1225
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1245..1262
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1272..2372
                     /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     2346..2369
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2427..2447
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2457..2477
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2487..2507
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2535..6638
                     /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             2535..6638
                     /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             6645..6665
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6672..6692
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6830..6848)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6844..6891
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7257..7262,7263..7279,7280..7285))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(7312..7330)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7398..7502
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7503..7571,7572..8363)
                     /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(7721..7740)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8534..9122
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9023..9042
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9276..9293
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    9308..9447
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(9373..9392)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9533..9555
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(9549..9740)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc
       61 gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt
      121 ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc
      181 gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg
      241 gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg
      301 acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc
      361 tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg
      421 atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac
      481 gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg
      541 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
      601 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
      661 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
      721 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
      781 ctcaagggca tcggtcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
      841 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
      901 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
      961 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     1021 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     1081 tcgatcccgc gaaattaata cgactcacta tagggagacc acaacggttt ccctctagtg
     1141 ccggctccgg agagctcttt aattaagcgg ccgccctgca ggactcgagt tctagaaata
     1201 attttgttta actttaagaa ggagatatac atatgaaatc ttctcaccat caccatcacc
     1261 atggttcttc tatgaaaatc gaagaaggta aactggtaat ctggattaac ggcgataaag
     1321 gctataacgg tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca
     1381 ccgttgagca tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg
     1441 gccctgacat tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt
     1501 tggctgaaat caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg
     1561 ccgtacgtta caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga
     1621 tttataacaa agatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg
     1681 ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact
     1741 tcacctggcc gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt
     1801 acgacattaa agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg
     1861 ttgacctgat taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg
     1921 cctttaataa aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg
     1981 acaccagcaa agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca
     2041 aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg
     2101 caaaagagtt cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag
     2161 acaaaccgct gggtgccgta gcgctgaagt cttacgagga agagttggcg aaagatccac
     2221 gtattgccgc cactatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga
     2281 tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga
     2341 ctgtcgatga agccctgaaa gacgcgcaga ctaattcgag ctcgaacaac aacaacaata
     2401 acaataacaa caacctcggg atcgaggaaa acctgtactt ccaatccaat gccactccaa
     2461 agaagaaacg gaaagtaggc ggctccccca agaagaagcg gaaggtaggt atccacggag
     2521 tcccagcagc taccatggat aagaaatact caataggctt agatatcggc acaaatagcg
     2581 tcggatgggc ggtgatcact gatgaatata aggttccgtc taaaaagttc aaggttctgg
     2641 gaaatacaga ccgccacagt atcaaaaaaa atcttatagg ggctctttta tttgacagtg
     2701 gagagacagc ggaagcgact cgtctcaaac ggacagctcg tagaaggtat acacgtcgga
     2761 agaatcgtat ttgttatcta caggagattt tttcaaatga gatggcgaaa gtagatgata
     2821 gtttctttca tcgacttgaa gagtcttttt tggtggaaga agacaagaag catgaacgtc
     2881 atcctatttt tggaaatata gtagatgaag ttgcttatca tgagaaatat ccaactatct
     2941 atcatctgcg aaaaaaattg gtagattcta ctgataaagc ggatttgcgc ttaatctatt
     3001 tggccttagc gcatatgatt aagtttcgtg gtcatttttt gattgaggga gatttaaatc
     3061 ctgataatag tgatgtggac aaactattta tccagttggt acaaacctac aatcaattat
     3121 ttgaagaaaa ccctattaac gcaagtggag tagatgctaa agcgattctt tctgcacgat
     3181 tgagtaaatc aagacgatta gaaaatctca ttgctcagct ccccggtgag aagaaaaatg
     3241 gcttatttgg gaatctcatt gctttgtcat tgggtttgac ccctaatttt aaatcaaatt
     3301 ttgatttggc agaagatgct aaattacagc tttcaaaaga tacttacgat gatgatttag
     3361 ataatttatt ggcgcaaatt ggagatcaat atgctgattt gtttttggca gctaagaatt
     3421 tatcagatgc tattttactt tcagatatcc taagagtaaa tactgaaata actaaggctc
     3481 ccctatcagc ttcaatgatt aaacgctacg atgaacatca tcaagacttg actcttttaa
     3541 aagctttagt tcgacaacaa cttccagaaa agtataaaga aatctttttt gatcaatcaa
     3601 aaaacggata tgcaggttat attgatgggg gagctagcca agaagaattt tataaattta
     3661 tcaaaccaat tttagaaaaa atggatggta ctgaggaatt attggtgaaa ctaaatcgtg
     3721 aagatttgct gcgcaagcaa cggacctttg acaacggctc tattccccat caaattcact
     3781 tgggtgagct gcatgctatt ttgagaagac aagaagactt ttatccattt ttaaaagaca
     3841 atcgtgagaa gattgaaaaa atcttgactt ttcgaattcc ttattatgtt ggtccattgg
     3901 cgcgtggcaa tagtcgtttt gcatggatga ctcggaagtc tgaagaaaca attaccccat
     3961 ggaattttga agaagttgtc gataaaggtg cttcagctca atcatttatt gaacgcatga
     4021 caaactttga taaaaatctt ccaaatgaaa aagtactacc aaaacatagt ttgctttatg
     4081 agtattttac ggtttataac gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa
     4141 aaccagcatt tctttcaggt gaacagaaga aagccattgt tgatttactc ttcaaaacaa
     4201 atcgaaaagt aaccgttaag caattaaaag aagattattt caaaaaaata gaatgttttg
     4261 atagtgttga aatttcagga gttgaagata gatttaatgc ttcattaggt acctaccatg
     4321 atttgctaaa aattattaaa gataaagatt ttttggataa tgaagaaaat gaagatatct
     4381 tagaggatat tgttttaaca ttgaccttat ttgaagatag ggagatgatt gaggaaagac
     4441 ttaaaacata tgctcacctc tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt
     4501 atactggttg gggacgtttg tctcgaaaat tgattaatgg tattagggat aagcaatctg
     4561 gcaaaacaat attagatttt ttgaaatcag atggttttgc caatcgcaat tttatgcagc
     4621 tgatccatga tgatagtttg acatttaaag aagacattca aaaagcacaa gtgtctggac
     4681 aaggcgatag tttacatgaa catattgcaa atttagctgg tagccctgct attaaaaaag
     4741 gtattttaca gactgtaaaa gttgttgatg aattggtcaa agtaatgggg cggcataagc
     4801 cagaaaatat cgttattgaa atggcacgtg aaaatcagac aactcaaaag ggccagaaaa
     4861 attcgcgaga gcgtatgaaa cgaatcgaag aaggtatcaa agaattagga agtcagattc
     4921 ttaaagagca tcctgttgaa aatactcaat tgcaaaatga aaagctctat ctctattatc
     4981 tccaaaatgg aagagacatg tatgtggacc aagaattaga tattaatcgt ttaagtgatt
     5041 atgatgtcga tcacattgtt ccacaaagtt tccttaaaga cgattcaata gacaataagg
     5101 tcttaacgcg ttctgataaa aatcgtggta aatcggataa cgttccaagt gaagaagtag
     5161 tcaaaaagat gaaaaactat tggagacaac ttctaaacgc caagttaatc actcaacgta
     5221 agtttgataa tttaacgaaa gctgaacgtg gaggtttgag tgaacttgat aaagctggtt
     5281 ttatcaaacg ccaattggtt gaaactcgcc aaatcactaa gcatgtggca caaattttgg
     5341 atagtcgcat gaatactaaa tacgatgaaa atgataaact tattcgagag gttaaagtga
     5401 ttaccttaaa atctaaatta gtttctgact tccgaaaaga tttccaattc tataaagtac
     5461 gtgagattaa caattaccat catgcccatg atgcgtatct aaatgccgtc gttggaactg
     5521 ctttgattaa gaaatatcca aaacttgaat cggagtttgt ctatggtgat tataaagttt
     5581 atgatgttcg taaaatgatt gctaagtctg agcaagaaat aggcaaagca accgcaaaat
     5641 atttctttta ctctaatatc atgaacttct tcaaaacaga aattacactt gcaaatggag
     5701 agattcgcaa acgccctcta atcgaaacta atggggaaac tggagaaatt gtctgggata
     5761 aagggcgaga ttttgccaca gtgcgcaaag tattgtccat gccccaagtc aatattgtca
     5821 agaaaacaga agtacagaca ggcggattct ccaaggagtc aattttacca aaaagaaatt
     5881 cggacaagct tattgctcgt aaaaaagact gggatccaaa aaaatatggt ggttttgata
     5941 gtccaacggt agcttattca gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga
     6001 agttaaaatc cgttaaagag ttactaggga tcacaattat ggaaagaagt tcctttgaaa
     6061 aaaatccgat tgacttttta gaagctaaag gatataagga agttaaaaaa gacttaatca
     6121 ttaaactacc taaatatagt ctttttgagt tagaaaacgg tcgtaaacgg atgctggcta
     6181 gtgccggaga attacaaaaa ggaaatgagc tggctctgcc aagcaaatat gtgaattttt
     6241 tatatttagc tagtcattat gaaaagttga agggtagtcc agaagataac gaacaaaaac
     6301 aattgtttgt ggagcagcat aagcattatt tagatgagat tattgagcaa atcagtgaat
     6361 tttctaagcg tgttatttta gcagatgcca atttagataa agttcttagt gcatataaca
     6421 aacatagaga caaaccaata cgtgaacaag cagaaaatat tattcattta tttacgttga
     6481 cgaatcttgg agctcccgct gcttttaaat attttgatac aacaattgat cgtaaacgat
     6541 atacgtctac aaaagaagtt ttagatgcca ctcttatcca tcaatccatc actggtcttt
     6601 atgaaacacg cattgatttg agtcagctag gaggtgacgg gtcacctaag aaaaaacgaa
     6661 aagttgagga tcctaaaaag aaacgaaaag ttgataccgg ttaataacat tggaagtgga
     6721 taacggatcc gcgatcgcgg cgcgccacct ggtggccggc cggtaccacg cgtgcgcgct
     6781 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     6841 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     6901 ggaactatat ccggatatcc acaggacggg tgtggtcgcc atgatcgcgt agtcgatagt
     6961 ggctccaagt agcgaagcga gcaggactgg gcggcggcca aagcggtcgg acagtgctcc
     7021 gagaacgggt gcgcatagaa attgcatcaa cgcatatagc gctagcagca cgccatagtg
     7081 actggcgatg ctgtcggaat ggacgatatc ccgcaagagg cccggcagta ccggcataac
     7141 caagcctatg cctacagcat ccagggtgac ggtgccgagg atgacgatga gcgcattgtt
     7201 agatttcata cacggtgcct gactgcgtta gcaatttaac tgtgataaac taccgcatta
     7261 aagcttatcg atgataagct gtcaaacatg agaattcttg aagacgaaag ggcctcgtga
     7321 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     7381 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     7441 tgtatccgct catgagacaa taaccctgat aaatgcttca ataacattga aaaaggaaga
     7501 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     7561 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     7621 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     7681 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     7741 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     7801 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     7861 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     7921 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     7981 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     8041 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     8101 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     8161 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     8221 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     8281 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     8341 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     8401 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     8461 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     8521 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     8581 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     8641 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     8701 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     8761 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     8821 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     8881 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     8941 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     9001 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     9061 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     9121 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     9181 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     9241 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     9301 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     9361 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     9421 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     9481 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     9541 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     9601 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     9661 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     9721 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     9781 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     9841 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     9901 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     9961 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
    10021 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
    10081 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
    10141 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
    10201 acccg
//
