LOCUS       sequence-177330-       10175 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 10175)
  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 10175)
  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: 1xNLS-pMJ915v2
FEATURES             Location/Qualifiers
     source          1..10175
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     103..120
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    135..274
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(200..219)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     360..382
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(376..567)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2025..2044
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2128..2147
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2128..2146
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2235..2257
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2277..2294
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2304..3404
                     /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     3378..3401
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3459..3479
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3489..3509
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3537..7640
                     /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             3537..7640
                     /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             7647..7667
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7674..7694
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(7832..7850)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7846..7893
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(8259..8264,8265..8281,8282..8287))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(8314..8332)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8400..8504
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8505..8573,8574..9365)
                     /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(8723..8742)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9536..10124
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10025..10044
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
       61 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
      121 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
      181 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
      241 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
      301 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
      361 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
      421 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
      481 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
      541 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
      601 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
      661 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
      721 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
      781 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
      841 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
      901 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
      961 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     1021 catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga
     1081 gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg
     1141 caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct
     1201 tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca
     1261 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca
     1321 taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc
     1381 gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac
     1441 gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct
     1501 cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg
     1561 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     1621 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     1681 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     1741 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     1801 gggttgaagg ctctcaaggg catcggtcga cgctctccct tatgcgactc ctgcattagg
     1861 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     1921 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     1981 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     2041 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     2101 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tatagggaga ccacaacggt
     2161 ttccctctag tgccggctcc ggagagctct ttaattaagc ggccgccctg caggactcga
     2221 gttctagaaa taattttgtt taactttaag aaggagatat acatatgaaa tcttctcacc
     2281 atcaccatca ccatggttct tctatgaaaa tcgaagaagg taaactggta atctggatta
     2341 acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg
     2401 gaattaaagt caccgttgag catccggata aactggaaga gaaattccca caggttgcgg
     2461 caactggcga tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc
     2521 aatctggcct gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt
     2581 ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag
     2641 cgttatcgct gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga
     2701 tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc
     2761 aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg
     2821 aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc
     2881 tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca
     2941 tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat
     3001 ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg
     3061 gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga
     3121 acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag
     3181 cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg
     3241 cgaaagatcc acgtattgcc gccactatgg aaaacgccca gaaaggtgaa atcatgccga
     3301 acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca
     3361 gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactaattcg agctcgaaca
     3421 acaacaacaa taacaataac aacaacctcg ggatcgagga aaacctgtac ttccaatcca
     3481 atgccacccc aaagaagaaa cggaaagtag gcatccacgg agtcccagca gctaccatgg
     3541 ataagaaata ctcaataggc ttagatatcg gcacaaatag cgtcggatgg gcggtgatca
     3601 ctgatgaata taaggttccg tctaaaaagt tcaaggttct gggaaataca gaccgccaca
     3661 gtatcaaaaa aaatcttata ggggctcttt tatttgacag tggagagaca gcggaagcga
     3721 ctcgtctcaa acggacagct cgtagaaggt atacacgtcg gaagaatcgt atttgttatc
     3781 tacaggagat tttttcaaat gagatggcga aagtagatga tagtttcttt catcgacttg
     3841 aagagtcttt tttggtggaa gaagacaaga agcatgaacg tcatcctatt tttggaaata
     3901 tagtagatga agttgcttat catgagaaat atccaactat ctatcatctg cgaaaaaaat
     3961 tggtagattc tactgataaa gcggatttgc gcttaatcta tttggcctta gcgcatatga
     4021 ttaagtttcg tggtcatttt ttgattgagg gagatttaaa tcctgataat agtgatgtgg
     4081 acaaactatt tatccagttg gtacaaacct acaatcaatt atttgaagaa aaccctatta
     4141 acgcaagtgg agtagatgct aaagcgattc tttctgcacg attgagtaaa tcaagacgat
     4201 tagaaaatct cattgctcag ctccccggtg agaagaaaaa tggcttattt gggaatctca
     4261 ttgctttgtc attgggtttg acccctaatt ttaaatcaaa ttttgatttg gcagaagatg
     4321 ctaaattaca gctttcaaaa gatacttacg atgatgattt agataattta ttggcgcaaa
     4381 ttggagatca atatgctgat ttgtttttgg cagctaagaa tttatcagat gctattttac
     4441 tttcagatat cctaagagta aatactgaaa taactaaggc tcccctatca gcttcaatga
     4501 ttaaacgcta cgatgaacat catcaagact tgactctttt aaaagcttta gttcgacaac
     4561 aacttccaga aaagtataaa gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt
     4621 atattgatgg gggagctagc caagaagaat tttataaatt tatcaaacca attttagaaa
     4681 aaatggatgg tactgaggaa ttattggtga aactaaatcg tgaagatttg ctgcgcaagc
     4741 aacggacctt tgacaacggc tctattcccc atcaaattca cttgggtgag ctgcatgcta
     4801 ttttgagaag acaagaagac ttttatccat ttttaaaaga caatcgtgag aagattgaaa
     4861 aaatcttgac ttttcgaatt ccttattatg ttggtccatt ggcgcgtggc aatagtcgtt
     4921 ttgcatggat gactcggaag tctgaagaaa caattacccc atggaatttt gaagaagttg
     4981 tcgataaagg tgcttcagct caatcattta ttgaacgcat gacaaacttt gataaaaatc
     5041 ttccaaatga aaaagtacta ccaaaacata gtttgcttta tgagtatttt acggtttata
     5101 acgaattgac aaaggtcaaa tatgttactg aaggaatgcg aaaaccagca tttctttcag
     5161 gtgaacagaa gaaagccatt gttgatttac tcttcaaaac aaatcgaaaa gtaaccgtta
     5221 agcaattaaa agaagattat ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag
     5281 gagttgaaga tagatttaat gcttcattag gtacctacca tgatttgcta aaaattatta
     5341 aagataaaga ttttttggat aatgaagaaa atgaagatat cttagaggat attgttttaa
     5401 cattgacctt atttgaagat agggagatga ttgaggaaag acttaaaaca tatgctcacc
     5461 tctttgatga taaggtgatg aaacagctta aacgtcgccg ttatactggt tggggacgtt
     5521 tgtctcgaaa attgattaat ggtattaggg ataagcaatc tggcaaaaca atattagatt
     5581 ttttgaaatc agatggtttt gccaatcgca attttatgca gctgatccat gatgatagtt
     5641 tgacatttaa agaagacatt caaaaagcac aagtgtctgg acaaggcgat agtttacatg
     5701 aacatattgc aaatttagct ggtagccctg ctattaaaaa aggtatttta cagactgtaa
     5761 aagttgttga tgaattggtc aaagtaatgg ggcggcataa gccagaaaat atcgttattg
     5821 aaatggcacg tgaaaatcag acaactcaaa agggccagaa aaattcgcga gagcgtatga
     5881 aacgaatcga agaaggtatc aaagaattag gaagtcagat tcttaaagag catcctgttg
     5941 aaaatactca attgcaaaat gaaaagctct atctctatta tctccaaaat ggaagagaca
     6001 tgtatgtgga ccaagaatta gatattaatc gtttaagtga ttatgatgtc gatcacattg
     6061 ttccacaaag tttccttaaa gacgattcaa tagacaataa ggtcttaacg cgttctgata
     6121 aaaatcgtgg taaatcggat aacgttccaa gtgaagaagt agtcaaaaag atgaaaaact
     6181 attggagaca acttctaaac gccaagttaa tcactcaacg taagtttgat aatttaacga
     6241 aagctgaacg tggaggtttg agtgaacttg ataaagctgg ttttatcaaa cgccaattgg
     6301 ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt ggatagtcgc atgaatacta
     6361 aatacgatga aaatgataaa cttattcgag aggttaaagt gattacctta aaatctaaat
     6421 tagtttctga cttccgaaaa gatttccaat tctataaagt acgtgagatt aacaattacc
     6481 atcatgccca tgatgcgtat ctaaatgccg tcgttggaac tgctttgatt aagaaatatc
     6541 caaaacttga atcggagttt gtctatggtg attataaagt ttatgatgtt cgtaaaatga
     6601 ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa atatttcttt tactctaata
     6661 tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg agagattcgc aaacgccctc
     6721 taatcgaaac taatggggaa actggagaaa ttgtctggga taaagggcga gattttgcca
     6781 cagtgcgcaa agtattgtcc atgccccaag tcaatattgt caagaaaaca gaagtacaga
     6841 caggcggatt ctccaaggag tcaattttac caaaaagaaa ttcggacaag cttattgctc
     6901 gtaaaaaaga ctgggatcca aaaaaatatg gtggttttga tagtccaacg gtagcttatt
     6961 cagtcctagt ggttgctaag gtggaaaaag ggaaatcgaa gaagttaaaa tccgttaaag
     7021 agttactagg gatcacaatt atggaaagaa gttcctttga aaaaaatccg attgactttt
     7081 tagaagctaa aggatataag gaagttaaaa aagacttaat cattaaacta cctaaatata
     7141 gtctttttga gttagaaaac ggtcgtaaac ggatgctggc tagtgccgga gaattacaaa
     7201 aaggaaatga gctggctctg ccaagcaaat atgtgaattt tttatattta gctagtcatt
     7261 atgaaaagtt gaagggtagt ccagaagata acgaacaaaa acaattgttt gtggagcagc
     7321 ataagcatta tttagatgag attattgagc aaatcagtga attttctaag cgtgttattt
     7381 tagcagatgc caatttagat aaagttctta gtgcatataa caaacataga gacaaaccaa
     7441 tacgtgaaca agcagaaaat attattcatt tatttacgtt gacgaatctt ggagctcccg
     7501 ctgcttttaa atattttgat acaacaattg atcgtaaacg atatacgtct acaaaagaag
     7561 ttttagatgc cactcttatc catcaatcca tcactggtct ttatgaaaca cgcattgatt
     7621 tgagtcagct aggaggtgac gggtcaccta agaaaaaacg aaaagttgag gatcctaaaa
     7681 agaaacgaaa agttgatacc ggttaataac attggaagtg gataacggat ccgcgatcgc
     7741 ggcgcgccac ctggtggccg gccggtacca cgcgtgcgcg ctgatccggc tgctaacaaa
     7801 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     7861 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatat
     7921 ccacaggacg ggtgtggtcg ccatgatcgc gtagtcgata gtggctccaa gtagcgaagc
     7981 gagcaggact gggcggcggc caaagcggtc ggacagtgct ccgagaacgg gtgcgcatag
     8041 aaattgcatc aacgcatata gcgctagcag cacgccatag tgactggcga tgctgtcgga
     8101 atggacgata tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc
     8161 atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca tacacggtgc
     8221 ctgactgcgt tagcaattta actgtgataa actaccgcat taaagcttat cgatgataag
     8281 ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     8341 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     8401 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     8461 aataaccctg ataaatgctt caataacatt gaaaaaggaa gagtatgagt attcaacatt
     8521 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     8581 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     8641 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     8701 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     8761 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     8821 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     8881 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     8941 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     9001 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     9061 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     9121 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     9181 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     9241 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     9301 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     9361 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     9421 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     9481 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     9541 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     9601 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     9661 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     9721 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     9781 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     9841 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     9901 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     9961 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
    10021 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
    10081 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
    10141 cctttttacg gttcctggcc ttttgctggc ctttt
//
