LOCUS       sequence-275140-        9726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One plasmid encoding dual U6 promoter-driven sgRNAs and 
            mCherry-coupled Cas9-D10A nickase to enhance efficient and accurate 
            genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9726)
  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 9726)
  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-mCherry
FEATURES             Location/Qualifiers
     source          1..9726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        277..352
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        477..725
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        752..827
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(836..854)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(837..854)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        891..1176
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1178..1454
                     /label=chicken beta-actin promoter
     intron          1455..1682
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1694..1703
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1703..1768
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1775..1795
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1820..1843,1844..1846,1847..5920)
                     /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             1820..5920
                     /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             5921..5968
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5978..6034
                     /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             6035..6739
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(6177..6195)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(6767..6784)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6773..6980
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6989..7129
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6989..7118
                     /label=AAV2 ITR
     rep_origin      7204..7659
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7291..7310)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7501..7522
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7676..7695)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7795..7817
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7855..7873)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7941..8045
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8046..8114,8115..8906)
                     /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(8264..8283)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9077..9665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9566..9585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gggtcttcgg atccatgcga agacctgttt tagagctaga aatagcaagt
      301 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt
      361 tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat
      421 tctgcagaca aatggctcta gattaaccct cactaaaggg aaaggtcggg caggaagagg
      481 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
      541 ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
      601 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
      661 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
      721 acaccgggag accatcgatg agagggtctc agttttagag ctagaaatag caagttaaaa
      781 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttttctat
      841 agtgtcacct aaattgcagc tctggcccgt gtctcaaaat ctctggtacc cgttacataa
      901 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      961 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     1021 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     1081 ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt tcctacttgg
     1141 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
     1201 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
     1261 ttattttgtg cagcgatggg ggcggggggg agatggggcg cgcgccaggc ggggcggggc
     1321 ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg
     1381 cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa
     1441 gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg
     1501 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg
     1561 acggcccttc tcctccgggc tgtaattagc tgagcaagag gtaagggttt aagggatggt
     1621 tggttggtgg ggtattaatg tttaattacc tggagcacct gcctgaaatc actttttttc
     1681 aggttggacc ggtgccacca tggactataa ggaccacgac ggagactaca aggatcatga
     1741 tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat
     1801 ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggccatcg gcaccaactc
     1861 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct
     1921 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag
     1981 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg
     2041 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga
     2101 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg
     2161 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat
     2221 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta
     2281 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa
     2341 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct
     2401 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag
     2461 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa
     2521 tggcctgttc ggcaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa
     2581 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct
     2641 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa
     2701 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc
     2761 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct
     2821 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag
     2881 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt
     2941 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag
     3001 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca
     3061 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga
     3121 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct
     3181 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc
     3241 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat
     3301 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta
     3361 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag
     3421 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac
     3481 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt
     3541 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca
     3601 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat
     3661 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg
     3721 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag
     3781 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc
     3841 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca
     3901 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg
     3961 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa
     4021 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa
     4081 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa
     4141 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat
     4201 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta
     4261 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga
     4321 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa
     4381 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt
     4441 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag
     4501 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg
     4561 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct
     4621 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt
     4681 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt
     4741 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac
     4801 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt
     4861 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa
     4921 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg
     4981 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga
     5041 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt
     5101 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa
     5161 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga
     5221 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa
     5281 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga
     5341 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat
     5401 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc
     5461 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt
     5521 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa
     5581 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga
     5641 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa
     5701 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct
     5761 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag
     5821 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct
     5881 gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa
     5941 aaaggccggc caggcaaaaa agaaaaaggg ctccggagcc acgaacttct ctctgttaaa
     6001 gcaagcagga gacgtggaag aaaaccccgg tcctgtgagc aagggcgagg aggataacat
     6061 ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
     6121 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     6181 gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt
     6241 catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
     6301 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
     6361 gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     6421 cggcaccaac ttcccctccg acggccccgt aatgcagaaa aagaccatgg gctgggaggc
     6481 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct
     6541 gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa
     6601 gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa
     6661 cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg
     6721 catggacgag ctgtacaagg aattctaact agagctcgct gatcagcctc gactgtgcct
     6781 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     6841 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     6901 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag
     6961 aatagcaggc atgctgggga gcggccgcag gaacccctag tgatggagtt ggccactccc
     7021 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     7081 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     7141 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     7201 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     7261 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     7321 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     7381 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     7441 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     7501 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     7561 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     7621 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     7681 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     7741 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     7801 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     7861 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     7921 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     7981 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     8041 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     8101 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     8161 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     8221 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     8281 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     8341 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     8401 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     8461 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     8521 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     8581 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     8641 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     8701 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     8761 gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     8821 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     8881 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     8941 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     9001 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     9061 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     9121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     9181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     9241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     9301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     9361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     9421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     9481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     9541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     9601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     9661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     9721 acatgt
//
