LOCUS       sequence-373032-        9625 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One plasmid encoding dual U6 promoter-driven sgRNAs and 
            Cas9-D10A nickase linked via 2A peptide with puromycin resistant 
            marker to enhance efficient and accurate genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9625)
  AUTHORS   Chiang TW, le Sage C, Larrieu D, Demir M, Jackson SP
  TITLE     CRISPR-Cas9(D10A) nickase-based genotypic and phenotypic screening 
            to enhance genome editing.
  JOURNAL   Sci Rep. 2016 Apr 15;6:24356. doi: 10.1038/srep24356.
  PUBMED    27079678
REFERENCE   2  (bases 1 to 9625)
  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: "Sci Rep."; 
            date: "2016-04-15"; volume: "6:24356. doi"; pages: " 
            10.1038/srep24356"
COMMENT     Sequence Label: AIO-Puro
FEATURES             Location/Qualifiers
     source          1..9625
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..255
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        283..358
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        483..731
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        758..833
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(842..860)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(843..860)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        897..1182
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1184..1461
                     /label=chicken beta-actin promoter
     intron          1462..1689
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1701..1710
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1710..1775
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1782..1802
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1827..1850,1851..1853,1854..5927)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1827..5927
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5928..5975
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5991..6047
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             6048..6644
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     6541..6561
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6672..6689)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6678..6885
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6894..7034
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6894..7023
                     /label=AAV2 ITR
     rep_origin      7109..7564
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7196..7215)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7406..7427
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7581..7600)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7700..7722
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7760..7778)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7846..7950
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7951..8019,8020..8811)
                     /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(8169..8188)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8982..9570
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9471..9490
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acatgtgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
       61 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      121 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      181 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      241 aaaggacgaa acaccggggt cttcggatcc atgcgaagac ctgttttaga gctagaaata
      301 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
      361 ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgtttt tagcgcgtgc
      421 gccaattctg cagacaaatg gctctagatt aaccctcact aaagggaaag gtcgggcagg
      481 aagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
      541 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
      601 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
      661 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
      721 gacgaaacac cgggagacca tcgatgagag ggtctcagtt ttagagctag aaatagcaag
      781 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
      841 ttctatagtg tcacctaaat tgcagctctg gcccgtgtct caaaatctct ggtacccgtt
      901 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      961 tcaatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     1021 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     1081 aatgacggta aatggcccgc ctggcattgt gcccagtaca tgaccttatg ggactttcct
     1141 acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg
     1201 ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt
     1261 ttttaattat tttgtgcagc gatgggggcg gggggggggg gggggcgcgc gccrggsggg
     1321 gsggggsggg gsgrggggsg gggsggggsg aggcggagag gtgcggcggc agccaatcag
     1381 agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa
     1441 aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc
     1501 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc
     1561 gggcgggacg gcccttctcc tccgggctgt aattagctga gcaagaggta agggtttaag
     1621 ggatggttgg ttggtggggt attaatgttt aattacctgg agcacctgcc tgaaatcact
     1681 ttttttcagg ttggaccggt gccaccatgg actataagga ccacgacgga gactacaagg
     1741 atcatgatat tgattacaaa gacgatgacg ataagatggc cccaaagaag aagcggaagg
     1801 tcggtatcca cggagtccca gcagccgaca agaagtacag catcggcctg gccatcggca
     1861 ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca
     1921 aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt
     1981 tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca
     2041 ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg
     2101 tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc
     2161 acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc
     2221 ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc
     2281 tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg
     2341 acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca
     2401 accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt
     2461 ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga
     2521 agaagaatgg cctgttcggc aacctgattg ccctgagcct gggcctgacc cccaacttca
     2581 agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg
     2641 acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg
     2701 ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca
     2761 ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga
     2821 ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg
     2881 accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct
     2941 acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc
     3001 tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc
     3061 agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc
     3121 tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg
     3181 gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca
     3241 tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg
     3301 agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc
     3361 tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg
     3421 gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt
     3481 tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg
     3541 agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca
     3601 cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg
     3661 aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg
     3721 aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc
     3781 ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca
     3841 agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact
     3901 tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg
     3961 tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca
     4021 ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc
     4081 ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg
     4141 gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca
     4201 gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc
     4261 tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc
     4321 tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg
     4381 acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg
     4441 aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta
     4501 cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata
     4561 aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac
     4621 agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag
     4681 tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt
     4741 acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg
     4801 tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact
     4861 acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta
     4921 ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg
     4981 ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg
     5041 tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga
     5101 atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca
     5161 agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg
     5221 gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca
     5281 agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca
     5341 gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg
     5401 acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa
     5461 tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg
     5521 tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg
     5581 agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga
     5641 tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg
     5701 cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt
     5761 ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc
     5821 ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca
     5881 ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacaaa aggccggcgg
     5941 ccacgaaaaa ggccggccag gcaaaaaaga aaaaggaatt cggctccgga gcaacaaact
     6001 tctctctgct gaaacaagcc ggagatgtcg aagagaatcc tggaccgacc gagtacaagc
     6061 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     6121 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     6181 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     6241 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     6301 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     6361 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     6421 ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     6481 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggaaacc tccgcgcccc
     6541 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     6601 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgagaattc taactagagc
     6661 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     6721 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     6781 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     6841 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc
     6901 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     6961 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     7021 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     7081 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     7141 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     7201 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     7261 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     7321 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     7381 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     7441 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     7501 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     7561 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     7621 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     7681 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     7741 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     7801 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     7861 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     7921 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     7981 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     8041 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     8101 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     8161 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     8221 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     8281 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     8341 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     8401 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     8461 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     8521 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     8581 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     8641 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     8701 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     8761 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     8821 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     8881 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     8941 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     9001 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     9061 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     9121 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     9181 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     9241 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     9301 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     9361 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     9421 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     9481 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     9541 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     9601 ctggcctttt gctggccttt tgctc
//
