LOCUS       sequence-456946-        9171 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 from S. pyogenes with 2A-Puro, and cloning backbone for sgRNA 
            (V2.0).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9171)
  AUTHORS   Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F
  TITLE     Genome engineering using the CRISPR-Cas9 system.
  JOURNAL   Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. 
            Epub 2013 Oct 24.
  PUBMED    24157548
REFERENCE   2  (bases 1 to 9171)
  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"; doi: 
            "10.1038/nprot.2013.143"; journalName: "Nat Protoc"; date: 
            "2013-11"; volume: "8"; issue: "11"; pages: "2281-308"
COMMENT     Sequence Label: pSpCas9(BB)-2A-Puro (PX459) V2.0
FEATURES             Location/Qualifiers
     source          1..9171
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..286
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          563..790
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      802..811
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             811..876
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             883..903
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             928..5028
                     /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             928..5028
                     /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             5029..5076
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5092..5145
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5146..5745
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5146..5165)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5642..5662
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5773..5790)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5779..5986
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5995..6135
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5995..6124
                     /label=AAV2 ITR
     rep_origin      6210..6665
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6297..6316)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6507..6528
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6682..6701)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6801..6823
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6861..6879)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6947..7051
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7052..7120,7121..7912)
                     /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     complement(7270..7289)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8083..8671
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8572..8591
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8733..8981
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8733..8753
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8904..8923
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        9000..9075
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      121 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      181 cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt
      241 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      301 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      361 tattttttaa ttattttgtg cagcgatggg ggcggggggg ggggggggcg cgccrggggg
      421 gggggggggg gggggggggg gggggggggs grggsggrgr ggkgsggcgg cagccaatca
      481 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
      541 aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc
      601 cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag
      661 cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt aagggtttaa
      721 gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc ctgaaatcac
      781 tttttttcag gttggaccgg tgccaccatg gactataagg accacgacgg agactacaag
      841 gatcatgata ttgattacaa agacgatgac gataagatgg ccccaaagaa gaagcggaag
      901 gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct ggacatcggc
      961 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc
     1021 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg
     1081 ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac
     1141 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag
     1201 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag
     1261 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac
     1321 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg
     1381 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc
     1441 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac
     1501 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg
     1561 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag
     1621 aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc
     1681 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac
     1741 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc
     1801 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc
     1861 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg
     1921 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc
     1981 gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc
     2041 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag
     2101 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac
     2161 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc
     2221 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg
     2281 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc
     2341 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc
     2401 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc
     2461 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag
     2521 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg
     2581 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc
     2641 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc
     2701 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac
     2761 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc
     2821 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag
     2881 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac
     2941 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac
     3001 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag
     3061 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc
     3121 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc
     3181 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag
     3241 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc
     3301 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac
     3361 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg
     3421 ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc
     3481 gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc
     3541 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt
     3601 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat
     3661 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca
     3721 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa
     3781 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt
     3841 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc
     3901 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac
     3961 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct
     4021 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
     4081 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc
     4141 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg
     4201 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
     4261 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
     4321 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
     4381 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc
     4441 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
     4501 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
     4561 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
     4621 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
     4681 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag
     4741 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc
     4801 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg
     4861 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
     4921 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
     4981 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa aaggccggcg
     5041 gccacgaaaa aggccggcca ggcaaaaaag aaaaaggaat tcggcagtgg agagggcaga
     5101 ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaatgac cgagtacaag
     5161 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     5221 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     5281 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     5341 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     5401 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     5461 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     5521 gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     5581 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     5641 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     5701 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgagaatt ctaactagag
     5761 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     5821 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     5881 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     5941 acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac
     6001 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     6061 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     6121 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     6181 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     6241 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     6301 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     6361 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     6421 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     6481 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     6541 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     6601 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     6661 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     6721 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     6781 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     6841 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     6901 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     6961 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     7021 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     7081 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     7141 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     7201 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     7261 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     7321 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     7381 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     7441 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     7501 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     7561 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     7621 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     7681 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     7741 tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga
     7801 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     7861 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     7921 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     7981 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     8041 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     8101 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     8161 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     8221 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     8281 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     8341 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     8401 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     8461 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     8521 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     8581 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     8641 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     8701 cctggccttt tgctggcctt ttgctcacat gtgagggcct atttcccatg attccttcat
     8761 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     8821 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     8881 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     8941 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgggtcttcg agaagacctg
     9001 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     9061 gcaccgagtc ggtgcttttt tgttttagag ctagaaatag caagttaaaa taaggctagt
     9121 ccgtttttag cgcgtgcgcc aattctgcag acaaatggct ctagaggtac c
//
