LOCUS       pMJ915       10121 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express Streptococcus pyogenes Cas9 carrying two C-terminal SV40 
            NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10121)
  AUTHORS   Lin S, Staahl BT, Alla RK, Doudna JA
  TITLE     Enhanced homology-directed human genome engineering by controlled 
            timing of CRISPR/Cas9 delivery.
  JOURNAL   Elife. 2014 Dec 15;3:e04766. doi: 10.7554/eLife.04766.
  PUBMED    25497837
REFERENCE   2  (bases 1 to 10121)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            //gt12365.cn
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2014-12-15"; volume: "3:e04766. doi"; pages: " 
            10.7554/eLife.04766"
COMMENT     Sequence Label: pMJ915
FEATURES             Location/Qualifiers
     source          1..10121
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1343..1362
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1446..1465
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1446..1464
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1553..1575
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1595..1612
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1622..2722
                     /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     2696..2719
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2777..2797
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     regulatory      2801..2810
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2807..6910
                     /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             2807..6910
                     /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             6917..6937
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6944..6964
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(7096..7114)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7110..7157
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7523..7528,7529..7545,7546..7551))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(7578..7596)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7664..7768
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7769..7837,7838..8629)
                     /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(7987..8006)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8800..9388
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9289..9308
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9542..9559
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    9574..9713
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(9639..9658)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9799..9821
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(9815..10006)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
       61 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
      121 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
      181 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
      241 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
      301 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac
      361 ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg
      421 ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt
      481 cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc
      541 ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg gcgcctacaa
      601 tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg acgatcagcg
      661 gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc tgtccctgat
      721 ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg atgccgccgg
      781 aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga
      841 cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
      901 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
      961 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     1021 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     1081 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     1141 tcggtcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     1201 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     1261 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     1321 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     1381 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     1441 gaaattaata cgactcacta tagggagacc acaacggttt ccctctagtg ccggctccgg
     1501 agagctcttt aattaagcgg ccgccctgca ggactcgagt tctagaaata attttgttta
     1561 actttaagaa ggagatatac atatgaaatc ttctcaccat caccatcacc atggttcttc
     1621 tatgaaaatc gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg
     1681 tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca
     1741 tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat
     1801 tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat
     1861 caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta
     1921 caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa
     1981 agatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact
     2041 gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc
     2101 gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa
     2161 agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat
     2221 taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa
     2281 aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa
     2341 agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt
     2401 tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt
     2461 cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct
     2521 gggtgccgta gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc
     2581 cactatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt
     2641 ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga
     2701 agccctgaaa gacgcgcaga ctaattcgag ctcgaacaac aacaacaata acaataacaa
     2761 caacctcggg atcgaggaaa acctgtactt ccaatccaat gccaccatgg ataagaaata
     2821 ctcaataggc ttagatatcg gcacaaatag cgtcggatgg gcggtgatca ctgatgaata
     2881 taaggttccg tctaaaaagt tcaaggttct gggaaataca gaccgccaca gtatcaaaaa
     2941 aaatcttata ggggctcttt tatttgacag tggagagaca gcggaagcga ctcgtctcaa
     3001 acggacagct cgtagaaggt atacacgtcg gaagaatcgt atttgttatc tacaggagat
     3061 tttttcaaat gagatggcga aagtagatga tagtttcttt catcgacttg aagagtcttt
     3121 tttggtggaa gaagacaaga agcatgaacg tcatcctatt tttggaaata tagtagatga
     3181 agttgcttat catgagaaat atccaactat ctatcatctg cgaaaaaaat tggtagattc
     3241 tactgataaa gcggatttgc gcttaatcta tttggcctta gcgcatatga ttaagtttcg
     3301 tggtcatttt ttgattgagg gagatttaaa tcctgataat agtgatgtgg acaaactatt
     3361 tatccagttg gtacaaacct acaatcaatt atttgaagaa aaccctatta acgcaagtgg
     3421 agtagatgct aaagcgattc tttctgcacg attgagtaaa tcaagacgat tagaaaatct
     3481 cattgctcag ctccccggtg agaagaaaaa tggcttattt gggaatctca ttgctttgtc
     3541 attgggtttg acccctaatt ttaaatcaaa ttttgatttg gcagaagatg ctaaattaca
     3601 gctttcaaaa gatacttacg atgatgattt agataattta ttggcgcaaa ttggagatca
     3661 atatgctgat ttgtttttgg cagctaagaa tttatcagat gctattttac tttcagatat
     3721 cctaagagta aatactgaaa taactaaggc tcccctatca gcttcaatga ttaaacgcta
     3781 cgatgaacat catcaagact tgactctttt aaaagcttta gttcgacaac aacttccaga
     3841 aaagtataaa gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt atattgatgg
     3901 gggagctagc caagaagaat tttataaatt tatcaaacca attttagaaa aaatggatgg
     3961 tactgaggaa ttattggtga aactaaatcg tgaagatttg ctgcgcaagc aacggacctt
     4021 tgacaacggc tctattcccc atcaaattca cttgggtgag ctgcatgcta ttttgagaag
     4081 acaagaagac ttttatccat ttttaaaaga caatcgtgag aagattgaaa aaatcttgac
     4141 ttttcgaatt ccttattatg ttggtccatt ggcgcgtggc aatagtcgtt ttgcatggat
     4201 gactcggaag tctgaagaaa caattacccc atggaatttt gaagaagttg tcgataaagg
     4261 tgcttcagct caatcattta ttgaacgcat gacaaacttt gataaaaatc ttccaaatga
     4321 aaaagtacta ccaaaacata gtttgcttta tgagtatttt acggtttata acgaattgac
     4381 aaaggtcaaa tatgttactg aaggaatgcg aaaaccagca tttctttcag gtgaacagaa
     4441 gaaagccatt gttgatttac tcttcaaaac aaatcgaaaa gtaaccgtta agcaattaaa
     4501 agaagattat ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag gagttgaaga
     4561 tagatttaat gcttcattag gtacctacca tgatttgcta aaaattatta aagataaaga
     4621 ttttttggat aatgaagaaa atgaagatat cttagaggat attgttttaa cattgacctt
     4681 atttgaagat agggagatga ttgaggaaag acttaaaaca tatgctcacc tctttgatga
     4741 taaggtgatg aaacagctta aacgtcgccg ttatactggt tggggacgtt tgtctcgaaa
     4801 attgattaat ggtattaggg ataagcaatc tggcaaaaca atattagatt ttttgaaatc
     4861 agatggtttt gccaatcgca attttatgca gctgatccat gatgatagtt tgacatttaa
     4921 agaagacatt caaaaagcac aagtgtctgg acaaggcgat agtttacatg aacatattgc
     4981 aaatttagct ggtagccctg ctattaaaaa aggtatttta cagactgtaa aagttgttga
     5041 tgaattggtc aaagtaatgg ggcggcataa gccagaaaat atcgttattg aaatggcacg
     5101 tgaaaatcag acaactcaaa agggccagaa aaattcgcga gagcgtatga aacgaatcga
     5161 agaaggtatc aaagaattag gaagtcagat tcttaaagag catcctgttg aaaatactca
     5221 attgcaaaat gaaaagctct atctctatta tctccaaaat ggaagagaca tgtatgtgga
     5281 ccaagaatta gatattaatc gtttaagtga ttatgatgtc gatcacattg ttccacaaag
     5341 tttccttaaa gacgattcaa tagacaataa ggtcttaacg cgttctgata aaaatcgtgg
     5401 taaatcggat aacgttccaa gtgaagaagt agtcaaaaag atgaaaaact attggagaca
     5461 acttctaaac gccaagttaa tcactcaacg taagtttgat aatttaacga aagctgaacg
     5521 tggaggtttg agtgaacttg ataaagctgg ttttatcaaa cgccaattgg ttgaaactcg
     5581 ccaaatcact aagcatgtgg cacaaatttt ggatagtcgc atgaatacta aatacgatga
     5641 aaatgataaa cttattcgag aggttaaagt gattacctta aaatctaaat tagtttctga
     5701 cttccgaaaa gatttccaat tctataaagt acgtgagatt aacaattacc atcatgccca
     5761 tgatgcgtat ctaaatgccg tcgttggaac tgctttgatt aagaaatatc caaaacttga
     5821 atcggagttt gtctatggtg attataaagt ttatgatgtt cgtaaaatga ttgctaagtc
     5881 tgagcaagaa ataggcaaag caaccgcaaa atatttcttt tactctaata tcatgaactt
     5941 cttcaaaaca gaaattacac ttgcaaatgg agagattcgc aaacgccctc taatcgaaac
     6001 taatggggaa actggagaaa ttgtctggga taaagggcga gattttgcca cagtgcgcaa
     6061 agtattgtcc atgccccaag tcaatattgt caagaaaaca gaagtacaga caggcggatt
     6121 ctccaaggag tcaattttac caaaaagaaa ttcggacaag cttattgctc gtaaaaaaga
     6181 ctgggatcca aaaaaatatg gtggttttga tagtccaacg gtagcttatt cagtcctagt
     6241 ggttgctaag gtggaaaaag ggaaatcgaa gaagttaaaa tccgttaaag agttactagg
     6301 gatcacaatt atggaaagaa gttcctttga aaaaaatccg attgactttt tagaagctaa
     6361 aggatataag gaagttaaaa aagacttaat cattaaacta cctaaatata gtctttttga
     6421 gttagaaaac ggtcgtaaac ggatgctggc tagtgccgga gaattacaaa aaggaaatga
     6481 gctggctctg ccaagcaaat atgtgaattt tttatattta gctagtcatt atgaaaagtt
     6541 gaagggtagt ccagaagata acgaacaaaa acaattgttt gtggagcagc ataagcatta
     6601 tttagatgag attattgagc aaatcagtga attttctaag cgtgttattt tagcagatgc
     6661 caatttagat aaagttctta gtgcatataa caaacataga gacaaaccaa tacgtgaaca
     6721 agcagaaaat attattcatt tatttacgtt gacgaatctt ggagctcccg ctgcttttaa
     6781 atattttgat acaacaattg atcgtaaacg atatacgtct acaaaagaag ttttagatgc
     6841 cactcttatc catcaatcca tcactggtct ttatgaaaca cgcattgatt tgagtcagct
     6901 aggaggtgac gggtcaccta agaaaaaacg aaaagttgag gatcctaaaa agaaacgaaa
     6961 agttgattaa taacattgga agtggataac ggatccgcga tcgcggcgcg ccacctggtg
     7021 gccggccggt accacgcgtg cgcgctgatc cggctgctaa caaagcccga aaggaagctg
     7081 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     7141 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatccacag gacgggtgtg
     7201 gtcgccatga tcgcgtagtc gatagtggct ccaagtagcg aagcgagcag gactgggcgg
     7261 cggccaaagc ggtcggacag tgctccgaga acgggtgcgc atagaaattg catcaacgca
     7321 tatagcgcta gcagcacgcc atagtgactg gcgatgctgt cggaatggac gatatcccgc
     7381 aagaggcccg gcagtaccgg cataaccaag cctatgccta cagcatccag ggtgacggtg
     7441 ccgaggatga cgatgagcgc attgttagat ttcatacacg gtgcctgact gcgttagcaa
     7501 tttaactgtg ataaactacc gcattaaagc ttatcgatga taagctgtca aacatgagaa
     7561 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     7621 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     7681 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     7741 gcttcaataa cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     7801 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     7861 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     7921 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     7981 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
     8041 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     8101 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     8161 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     8221 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     8281 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
     8341 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     8401 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     8461 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     8521 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     8581 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     8641 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     8701 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     8761 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     8821 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     8881 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     8941 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     9001 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     9061 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     9121 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     9181 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     9241 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     9301 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     9361 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     9421 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     9481 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     9541 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     9601 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     9661 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     9721 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     9781 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     9841 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     9901 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     9961 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
    10021 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
    10081 tacgggttac tgatgatgaa catgcccggt tactggaacg t
//