LOCUS       sequence-177336-       10265 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 10265)
  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 10265)
  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: 4xNLS-pMJ915v2
FEATURES             Location/Qualifiers
     source          1..10265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     6..28
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(22..213)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     1671..1690
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1774..1793
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1774..1792
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1881..1903
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1923..1940
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1950..3050
                     /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     3024..3047
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3105..3125
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3135..3155
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3165..3185
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3195..3215
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3225..3245
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3273..7376
                     /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             3273..7376
                     /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             7383..7403
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7410..7430
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(7568..7586)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7582..7629
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7995..8000,8001..8017,8018..8023))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(8050..8068)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8136..8240
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8241..8309,8310..9101)
                     /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(8459..8478)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9272..9860
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9761..9780
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10014..10031
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    10046..10185
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(10111..10130)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
       61 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
      121 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
      181 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
      241 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
      301 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
      361 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
      421 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
      481 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
      541 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
      601 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
      661 cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct
      721 gctggagatg gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt
      781 tctccgcaag aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg
      841 ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg
      901 cagacaaggt atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag
      961 gcggcataaa tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc
     1021 gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc
     1081 tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc
     1141 cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg
     1201 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     1261 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     1321 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     1381 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     1441 ctgactgggt tgaaggctct caagggcatc ggtcgacgct ctcccttatg cgactcctgc
     1501 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     1561 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     1621 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     1681 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     1741 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata gggagaccac
     1801 aacggtttcc ctctagtgcc ggctccggag agctctttaa ttaagcggcc gccctgcagg
     1861 actcgagttc tagaaataat tttgtttaac tttaagaagg agatatacat atgaaatctt
     1921 ctcaccatca ccatcaccat ggttcttcta tgaaaatcga agaaggtaaa ctggtaatct
     1981 ggattaacgg cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag
     2041 ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg
     2101 ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct
     2161 acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt
     2221 atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg
     2281 ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca aaaacctggg
     2341 aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca
     2401 acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca
     2461 agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct ggcgcgaaag
     2521 cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca gacaccgatt
     2581 actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc aacggcccgt
     2641 gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta ctgccgacct
     2701 tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca
     2761 gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact gatgaaggtc
     2821 tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag
     2881 agttggcgaa agatccacgt attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca
     2941 tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg
     3001 ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact aattcgagct
     3061 cgaacaacaa caacaataac aataacaaca acctcgggat cgaggaaaac ctgtacttcc
     3121 aatccaatgc cactcccaag aagaagcgaa aggtgggtgg gtccccaaag aagaaacgga
     3181 aagtaggcgg ctcccccaaa aagaagcgaa aagtaggggg tagccccaag aagaagcgga
     3241 aggtaggtat ccacggagtc ccagcagcta ccatggataa gaaatactca ataggcttag
     3301 atatcggcac aaatagcgtc ggatgggcgg tgatcactga tgaatataag gttccgtcta
     3361 aaaagttcaa ggttctggga aatacagacc gccacagtat caaaaaaaat cttatagggg
     3421 ctcttttatt tgacagtgga gagacagcgg aagcgactcg tctcaaacgg acagctcgta
     3481 gaaggtatac acgtcggaag aatcgtattt gttatctaca ggagattttt tcaaatgaga
     3541 tggcgaaagt agatgatagt ttctttcatc gacttgaaga gtcttttttg gtggaagaag
     3601 acaagaagca tgaacgtcat cctatttttg gaaatatagt agatgaagtt gcttatcatg
     3661 agaaatatcc aactatctat catctgcgaa aaaaattggt agattctact gataaagcgg
     3721 atttgcgctt aatctatttg gccttagcgc atatgattaa gtttcgtggt cattttttga
     3781 ttgagggaga tttaaatcct gataatagtg atgtggacaa actatttatc cagttggtac
     3841 aaacctacaa tcaattattt gaagaaaacc ctattaacgc aagtggagta gatgctaaag
     3901 cgattctttc tgcacgattg agtaaatcaa gacgattaga aaatctcatt gctcagctcc
     3961 ccggtgagaa gaaaaatggc ttatttggga atctcattgc tttgtcattg ggtttgaccc
     4021 ctaattttaa atcaaatttt gatttggcag aagatgctaa attacagctt tcaaaagata
     4081 cttacgatga tgatttagat aatttattgg cgcaaattgg agatcaatat gctgatttgt
     4141 ttttggcagc taagaattta tcagatgcta ttttactttc agatatccta agagtaaata
     4201 ctgaaataac taaggctccc ctatcagctt caatgattaa acgctacgat gaacatcatc
     4261 aagacttgac tcttttaaaa gctttagttc gacaacaact tccagaaaag tataaagaaa
     4321 tcttttttga tcaatcaaaa aacggatatg caggttatat tgatggggga gctagccaag
     4381 aagaatttta taaatttatc aaaccaattt tagaaaaaat ggatggtact gaggaattat
     4441 tggtgaaact aaatcgtgaa gatttgctgc gcaagcaacg gacctttgac aacggctcta
     4501 ttccccatca aattcacttg ggtgagctgc atgctatttt gagaagacaa gaagactttt
     4561 atccattttt aaaagacaat cgtgagaaga ttgaaaaaat cttgactttt cgaattcctt
     4621 attatgttgg tccattggcg cgtggcaata gtcgttttgc atggatgact cggaagtctg
     4681 aagaaacaat taccccatgg aattttgaag aagttgtcga taaaggtgct tcagctcaat
     4741 catttattga acgcatgaca aactttgata aaaatcttcc aaatgaaaaa gtactaccaa
     4801 aacatagttt gctttatgag tattttacgg tttataacga attgacaaag gtcaaatatg
     4861 ttactgaagg aatgcgaaaa ccagcatttc tttcaggtga acagaagaaa gccattgttg
     4921 atttactctt caaaacaaat cgaaaagtaa ccgttaagca attaaaagaa gattatttca
     4981 aaaaaataga atgttttgat agtgttgaaa tttcaggagt tgaagataga tttaatgctt
     5041 cattaggtac ctaccatgat ttgctaaaaa ttattaaaga taaagatttt ttggataatg
     5101 aagaaaatga agatatctta gaggatattg ttttaacatt gaccttattt gaagataggg
     5161 agatgattga ggaaagactt aaaacatatg ctcacctctt tgatgataag gtgatgaaac
     5221 agcttaaacg tcgccgttat actggttggg gacgtttgtc tcgaaaattg attaatggta
     5281 ttagggataa gcaatctggc aaaacaatat tagatttttt gaaatcagat ggttttgcca
     5341 atcgcaattt tatgcagctg atccatgatg atagtttgac atttaaagaa gacattcaaa
     5401 aagcacaagt gtctggacaa ggcgatagtt tacatgaaca tattgcaaat ttagctggta
     5461 gccctgctat taaaaaaggt attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag
     5521 taatggggcg gcataagcca gaaaatatcg ttattgaaat ggcacgtgaa aatcagacaa
     5581 ctcaaaaggg ccagaaaaat tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag
     5641 aattaggaag tcagattctt aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa
     5701 agctctatct ctattatctc caaaatggaa gagacatgta tgtggaccaa gaattagata
     5761 ttaatcgttt aagtgattat gatgtcgatc acattgttcc acaaagtttc cttaaagacg
     5821 attcaataga caataaggtc ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg
     5881 ttccaagtga agaagtagtc aaaaagatga aaaactattg gagacaactt ctaaacgcca
     5941 agttaatcac tcaacgtaag tttgataatt taacgaaagc tgaacgtgga ggtttgagtg
     6001 aacttgataa agctggtttt atcaaacgcc aattggttga aactcgccaa atcactaagc
     6061 atgtggcaca aattttggat agtcgcatga atactaaata cgatgaaaat gataaactta
     6121 ttcgagaggt taaagtgatt accttaaaat ctaaattagt ttctgacttc cgaaaagatt
     6181 tccaattcta taaagtacgt gagattaaca attaccatca tgcccatgat gcgtatctaa
     6241 atgccgtcgt tggaactgct ttgattaaga aatatccaaa acttgaatcg gagtttgtct
     6301 atggtgatta taaagtttat gatgttcgta aaatgattgc taagtctgag caagaaatag
     6361 gcaaagcaac cgcaaaatat ttcttttact ctaatatcat gaacttcttc aaaacagaaa
     6421 ttacacttgc aaatggagag attcgcaaac gccctctaat cgaaactaat ggggaaactg
     6481 gagaaattgt ctgggataaa gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc
     6541 cccaagtcaa tattgtcaag aaaacagaag tacagacagg cggattctcc aaggagtcaa
     6601 ttttaccaaa aagaaattcg gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa
     6661 aatatggtgg ttttgatagt ccaacggtag cttattcagt cctagtggtt gctaaggtgg
     6721 aaaaagggaa atcgaagaag ttaaaatccg ttaaagagtt actagggatc acaattatgg
     6781 aaagaagttc ctttgaaaaa aatccgattg actttttaga agctaaagga tataaggaag
     6841 ttaaaaaaga cttaatcatt aaactaccta aatatagtct ttttgagtta gaaaacggtc
     6901 gtaaacggat gctggctagt gccggagaat tacaaaaagg aaatgagctg gctctgccaa
     6961 gcaaatatgt gaatttttta tatttagcta gtcattatga aaagttgaag ggtagtccag
     7021 aagataacga acaaaaacaa ttgtttgtgg agcagcataa gcattattta gatgagatta
     7081 ttgagcaaat cagtgaattt tctaagcgtg ttattttagc agatgccaat ttagataaag
     7141 ttcttagtgc atataacaaa catagagaca aaccaatacg tgaacaagca gaaaatatta
     7201 ttcatttatt tacgttgacg aatcttggag ctcccgctgc ttttaaatat tttgatacaa
     7261 caattgatcg taaacgatat acgtctacaa aagaagtttt agatgccact cttatccatc
     7321 aatccatcac tggtctttat gaaacacgca ttgatttgag tcagctagga ggtgacgggt
     7381 cacctaagaa aaaacgaaaa gttgaggatc ctaaaaagaa acgaaaagtt gataccggtt
     7441 aataacattg gaagtggata acggatccgc gatcgcggcg cgccacctgg tggccggccg
     7501 gtaccacgcg tgcgcgctga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     7561 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     7621 ggttttttgc tgaaaggagg aactatatcc ggatatccac aggacgggtg tggtcgccat
     7681 gatcgcgtag tcgatagtgg ctccaagtag cgaagcgagc aggactgggc ggcggccaaa
     7741 gcggtcggac agtgctccga gaacgggtgc gcatagaaat tgcatcaacg catatagcgc
     7801 tagcagcacg ccatagtgac tggcgatgct gtcggaatgg acgatatccc gcaagaggcc
     7861 cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat
     7921 gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg
     7981 tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag aattcttgaa
     8041 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     8101 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     8161 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     8221 aacattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     8281 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     8341 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     8401 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     8461 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     8521 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     8581 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     8641 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     8701 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     8761 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     8821 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     8881 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     8941 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     9001 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     9061 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     9121 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     9181 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     9241 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     9301 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     9361 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     9421 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     9481 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     9541 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     9601 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     9661 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     9721 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     9781 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     9841 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     9901 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     9961 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
    10021 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
    10081 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
    10141 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
    10201 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
    10261 gtgac
//
