LOCUS       sequence-204720-       11834 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpCas9 fused to NmCas9 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11834)
  AUTHORS   Bolukbasi MF, Liu P, Luk K, Kwok SF, Gupta A, Amrani N, Sontheimer 
            EJ, Zhu LJ, Wolfe SA
  TITLE     Orthogonal Cas9-Cas9 chimeras provide a versatile platform for 
            genome editing.
  JOURNAL   Nat Commun. 2018 Nov 19;9(1):4856. doi: 10.1038/s41467-018-07310-x.
  PUBMED    30451839
REFERENCE   2  (bases 1 to 11834)
  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 
            Commun."; date: "2018-11-19"; volume: "9(1):4856. doi"; pages: " 
            10.1038/s41467-018-07310-x"
COMMENT     Sequence Label: pCSD_2XNLS-SpCas9WT-NLS-3xHA-2XNLS-NmCas9WT-2XNLS
FEATURES             Location/Qualifiers
     source          1..11834
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        33..51
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     33..50
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             101..148
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             167..187
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             194..4294
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             194..4294
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             194..4294
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4307..4327
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4328..4417
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             4430..4456
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             4472..4492
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4493..7735
                     /codon_start=1
                     /product="Cas9 endonuclease from the Neisseria meningitidis
                     Type II CRISPR/Cas system (Esvelt et al., 2013)"
                     /label=NmCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="AAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVF
                     ERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKS
                     LPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVAD
                     NAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEFG
                     NPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTK
                     LNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDN
                     AEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK
                     DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTE
                     EKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKE
                     IEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGR
                     LNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFK
                     ARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRV
                     FASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFD
                     GKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEK
                     LSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDL
                     EKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKT
                     GVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQL
                     IDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEG
                     IGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
     regulatory      7322..7331
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7748..7768
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7784..7831
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    7884..8018
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7908..7927)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7962..7981
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(8131..8151)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(8131..8149)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8170..8186)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8170..8186)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8183..8205)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8194..8210
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(8218..8224,8225..8242,8243..8248))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8263..8284
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8401..8418)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8572..9160)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8652..8671)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9331..10122,10123..10191))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     9954..9973
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10192..10296)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(10322..10777)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10459..10480)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     10671..10690
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        10848..11832
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 ccattctgcc tggggacgtc ggagcaagct tgatttaggt gacactatag aatacaagct
       61 acttgttctt tttgcaggat cccatcgatt cgaattcatg aaaaggccgg cggccacgaa
      121 aaaggccggc caggcaaaaa agaaaaagct ggacggcagc ggaagcccca agaagaagag
      181 gaaggtacca tgggacaaga agtactccat tgggctcgat atcggtacca acagcgtcgg
      241 ctgggccgtc attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa
      301 taccgatcgc cacagcataa agaagaacct cattggagcc ctcctgttcg actccgggga
      361 gacggccgaa gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa
      421 tcggatctgc tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt
      481 cttccatagg ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc
      541 aatctttggc aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca
      601 tctgaggaag aagctggtag acagtactga taaggctgac ttgcggttga tctatctcgc
      661 gctggcgcac atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga
      721 caacagcgat gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga
      781 ggagaacccg atcaacgcat ccggcgttga cgccaaagca atcctgagcg ctaggctgtc
      841 caaatcccgg cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct
      901 gtttggtaat cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga
      961 cctggccgaa gatgccaagc tgcaactgag caaagacacc tacgatgatg atctcgacaa
     1021 tctgctggcc cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc
     1081 agacgccatt ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct
     1141 gagcgctagt atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc
     1201 ccttgtcaga cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa
     1261 tggctacgcc ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa
     1321 gcccatcttg gaaaaaatgg acggcaccga ggagctgctg gtaaagctga acagagaaga
     1381 tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg
     1441 cgaactgcac gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag
     1501 ggaaaagatt gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgctcg
     1561 gggaaattcc agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa
     1621 cttcgaggaa gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa
     1681 ctttgataaa aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta
     1741 cttcacagtt tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc
     1801 agcattcctg tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg
     1861 gaaagttacc gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc
     1921 tgttgaaatc agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct
     1981 cctgaaaatc attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga
     2041 ggacattgtc ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa
     2101 aacttacgct catctcttcg acgacaaagt catgaaacag ctcaagagac gccgatatac
     2161 aggatggggg cggctgtcaa gaaaactgat caatggcatc cgagacaagc agagtggaaa
     2221 gacaatcctg gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat
     2281 ccatgatgac tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg
     2341 ggacagtctt cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat
     2401 actgcagacc gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga
     2461 gaatatcgtt atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag
     2521 tagggaaagg atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa
     2581 ggaacaccca gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca
     2641 gaacggcagg gacatgtacg tggatcagga actggacatc aaccggttgt ccgactacga
     2701 cgtggatcat atcgtgcccc aaagctttct caaagatgat tctattgata ataaagtgtt
     2761 gacaagatcc gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa
     2821 gaaaatgaaa aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt
     2881 cgataatctg actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat
     2941 caaaaggcag cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc
     3001 acgcatgaac accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac
     3061 tctgaagtct aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga
     3121 gatcaacaat taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact
     3181 tatcaaaaaa tatcccaagc tggaatctga atttgtttac ggagactata aagtgtacga
     3241 tgttaggaaa atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt
     3301 cttttacagc aatattatga attttttcaa gaccgagatt acactggcca atggagagat
     3361 tcggaagcga ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg
     3421 tagggatttc gcgacagtcc gcaaggtcct gtccatgccg caggtgaaca tcgttaaaaa
     3481 gaccgaagta cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga
     3541 caagctgatc gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc
     3601 tacagtcgct tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact
     3661 caaaagcgtc aaggaactgc tgggcatcac aatcatggag cgatccagct tcgagaaaaa
     3721 ccccatcgac tttctcgaag cgaaaggata taaagaggtc aaaaaagacc tcatcattaa
     3781 gctgcccaag tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc
     3841 gggcgagctg cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta
     3901 tctggccagc cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct
     3961 gttcgtggaa caacacaaac actaccttga tgagatcatc gagcaaataa gcgagttctc
     4021 caaaagagtg atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca
     4081 cagggataag cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa
     4141 cttgggcgcg cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac
     4201 ctctacaaag gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga
     4261 aacaagaatc gacctctctc agctcggtgg agacggcacc ggcgggccca agaagaagag
     4321 gaaggtatac ccatacgatg ttcctgacta tgcgggctat ccctatgacg tcccggacta
     4381 tgcaggatcg tatccttatg acgttccaga ttacgctgga tccgccgctc cggcagctaa
     4441 gaaaaagaaa ctggatttcg aatccggaat gcccaagaag aagaggaagg tggccgcctt
     4501 caagcccaac cccatcaact acatcctggg cctggacatc ggcatcgcca gcgtgggctg
     4561 ggccatggtg gagatcgacg aggacgagaa ccccatctgc ctgatcgacc tgggtgtgcg
     4621 cgtgttcgag cgcgctgagg tgcccaagac tggtgacagt ctggctatgg ctcgccggct
     4681 tgctcgctct gttcggcgcc ttactcgccg gcgcgctcac cgccttctgc gcgctcgccg
     4741 cctgctgaag cgcgagggtg tgctgcaggc tgccgacttc gacgagaacg gcctgatcaa
     4801 gagcctgccc aacactcctt ggcagctgcg cgctgccgct ctggaccgca agctgactcc
     4861 tctggagtgg agcgccgtgc tgctgcacct gatcaagcac cgcggctacc tgagccagcg
     4921 caagaacgag ggcgagaccg ccgacaagga gctgggtgct ctgctgaagg gcgtggccga
     4981 caacgcccac gccctgcaga ctggtgactt ccgcactcct gctgagctgg ccctgaacaa
     5041 gttcgagaag gagagcggcc acatccgcaa ccagcgcggc gactacagcc acaccttcag
     5101 ccgcaaggac ctgcaggccg agctgatcct gctgttcgag aagcagaagg agttcggcaa
     5161 cccccacgtg agcggcggcc tgaaggaggg catcgagacc ctgctgatga cccagcgccc
     5221 cgccctgagc ggcgacgccg tgcagaagat gctgggccac tgcaccttcg agccagccga
     5281 gcccaaggcc gccaagaaca cctacaccgc cgagcgcttc atctggctga ccaagctgaa
     5341 caacctgcgc atcctggagc agggcagcga gcgccccctg accgacaccg agcgcgccac
     5401 cctgatggac gagccctacc gcaagagcaa gctgacctac gcccaggccc gcaagctgct
     5461 gggtctggag gacaccgcct tcttcaaggg cctgcgctac ggcaaggaca acgccgaggc
     5521 cagcaccctg atggagatga aggcctacca cgccatcagc cgcgccctgg agaaggaggg
     5581 cctgaaggac aagaagagtc ctctgaacct gagccccgag ctgcaggacg agatcggcac
     5641 cgccttcagc ctgttcaaga ccgacgagga catcaccggc cgcctgaagg accgcatcca
     5701 gcccgagatc ctggaggccc tgctgaagca catcagcttc gacaagttcg tgcagatcag
     5761 cctgaaggcc ctgcgccgca tcgtgcccct gatggagcag ggcaagcgct acgacgaggc
     5821 ctgcgccgag atctacggcg accactacgg caagaagaac accgaggaga agatctacct
     5881 gcctcctatc cccgccgacg agatccgcaa ccccgtggtg ctgcgcgccc tgagccaggc
     5941 ccgcaaggtg atcaacggcg tggtgcgccg ctacggcagc cccgcccgca tccacatcga
     6001 gaccgcccgc gaggtgggca agagcttcaa ggaccgcaag gagatcgaga agcgccagga
     6061 ggagaaccgc aaggaccgcg agaaggccgc cgccaagttc cgcgagtact tccccaactt
     6121 cgtgggcgag cccaagagca aggacatcct gaagctgcgc ctgtacgagc agcagcacgg
     6181 caagtgcctg tacagcggca aggagatcaa cctgggccgc ctgaacgaga agggctacgt
     6241 ggagatcgac catgccctgc ccttcagccg cacctgggac gacagcttca acaacaaggt
     6301 gctggtgctg ggcagcgaga atcagaacaa gggcaaccag accccctacg agtacttcaa
     6361 cggcaaggac aacagccgcg agtggcagga gttcaaggcc cgcgtggaga ccagccgctt
     6421 cccccgcagc aagaagcagc gcatcctgct gcagaagttc gacgaggacg gcttcaagga
     6481 gcgcaacctg aacgacaccc gctacgtgaa ccgcttcctg tgccagttcg tggccgaccg
     6541 catgcgcctg accggcaagg gcaagaagcg cgtgttcgcc agcaacggcc agatcaccaa
     6601 cctgctgcgc ggcttctggg gcctgcgcaa ggtgcgcgcc gagaacgacc gccaccacgc
     6661 cctggacgcc gtggtggtgg cctgcagcac cgtggccatg cagcagaaga tcacccgctt
     6721 cgtgcgctac aaggagatga acgccttcga cggtaaaacc atcgacaagg agaccggcga
     6781 ggtgctgcac cagaagaccc acttccccca gccctgggag ttcttcgccc aggaggtgat
     6841 gatccgcgtg ttcggcaagc ccgacggcaa gcccgagttc gaggaggccg acacccccga
     6901 gaagctgcgc accctgctgg ccgagaagct gagcagccgc cctgaggccg tgcacgagta
     6961 cgtgactcct ctgttcgtga gccgcgcccc caaccgcaag atgagcggtc agggtcacat
     7021 ggagaccgtg aagagcgcca agcgcctgga cgagggcgtg agcgtgctgc gcgtgcccct
     7081 gacccagctg aagctgaagg acctggagaa gatggtgaac cgcgagcgcg agcccaagct
     7141 gtacgaggcc ctgaaggccc gcctggaggc ccacaaggac gaccccgcca aggccttcgc
     7201 cgagcccttc tacaagtacg acaaggccgg caaccgcacc cagcaggtga aggccgtgcg
     7261 cgtggagcag gtgcagaaga ccggcgtgtg ggtgcgcaac cacaacggca tcgccgacaa
     7321 cgccaccatg gtgcgcgtgg acgtgttcga gaagggcgac aagtactacc tggtgcccat
     7381 ctacagctgg caggtggcca agggcatcct gcccgaccgc gccgtggtgc agggcaagga
     7441 cgaggaggac tggcagctga tcgacgacag cttcaacttc aagttcagcc tgcaccccaa
     7501 cgacctggtg gaggtgatca ccaagaaggc ccgcatgttc ggctacttcg ccagctgcca
     7561 ccgcggcacc ggcaacatca acatccgcat ccacgacctg gaccacaaga tcggcaagaa
     7621 cggcatcctg gagggcatcg gcgtgaagac cgccctgagc ttccagaagt accagatcga
     7681 cgagctgggc aaggagatcc gcccctgccg cctgaagaag cgccctcctg tgcgcagcag
     7741 ggctgacccc aagaagaaga ggaaggtgga ggccagcctc gagaaaaggc cggcggccac
     7801 gaaaaaggcc ggccaggcaa aaaagaaaaa gggcagttaa tgatctagta atgatctaga
     7861 actatagtga gtcgtattac gtagatccag acatgataag atacattgat gagtttggac
     7921 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     7981 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     8041 ttatgtttca ggttcagggg gaggtgtggg aggtttttta attcgcggcc gcggcgccaa
     8101 tgcattgggc ccggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     8161 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     8221 catacgagcc gggagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     8281 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     8341 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     8401 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     8461 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     8521 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     8581 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     8641 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     8701 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     8761 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     8821 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     8881 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     8941 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     9001 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     9061 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     9121 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     9181 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     9241 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     9301 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     9361 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     9421 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     9481 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     9541 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     9601 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     9661 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     9721 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     9781 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     9841 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     9901 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     9961 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
    10021 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
    10081 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
    10141 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
    10201 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
    10261 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
    10321 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
    10381 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
    10441 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
    10501 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
    10561 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
    10621 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
    10681 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
    10741 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
    10801 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagtc gaccgccaat
    10861 tcaatatggc gtatatggac tcatgccaat tcaatatggt ggatctggac ctgtgccaat
    10921 tcaatatggc gtatatggac tcgtgccaat tcaatatggt ggatctggac cccagccaat
    10981 tcaatatggc ggacttggca ccatgccaat tcaatatggc ggacctggca ctgtgccaac
    11041 tggggagggg tctacttggc acggtgccaa gtttgaggag gggtcttggc cctgtgccaa
    11101 gtccgccata ttgaattggc atggtgccaa taatggcggc catattggct atatgccagg
    11161 atcaatatat aggcaatatc caatatggcc ctatgccaat atggctattg gccaggttca
    11221 atactatgta ttggccctat gccatatagt attccatata tgggttttcc tattgacgta
    11281 gatagcccct cccaatgggc ggtcccatat accatatatg gggcttccta ataccgccca
    11341 tagccactcc cccattgacg tcaatggtct ctatatatgg tctttcctat tgacgtcata
    11401 tgggcggtcc tattgacgta tatggcgcct cccccattga cgtcaattac ggtaaatggc
    11461 ccgcctggct caatgcccat tgacgtcaat aggaccaccc accattgacg tcaatgggat
    11521 ggctcattgc ccattcatat ccgttctcac gccccctatt gacgtcaatg acggtaaatg
    11581 gcccacttgg cagtacatca atatctatta atagtaactt ggcaagtaca ttactattgg
    11641 aagtacgcca gggtacattg gcagtactcc cattgacgtc aatggcggta aatggcccgc
    11701 gatggctgcc aagtacatcc ccattgacgt caatggggag gggcaatgac gcaaatgggc
    11761 gttccattga cgtaaatggg cggtaggcgt gcctaatggg aggtctatat aagcaatgct
    11821 cgtttaggga accg
//
