LOCUS       sequence-224211-        9516 bp    DNA     linear   UNA 16-APR-2025
DEFINITION  All-in-One plasmid encoding dual U6 promoter-driven sgRNAs and 
            EGFP-coupled Cas9-D10A nickase to enhance efficient and accurate 
            genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 9516)
  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 9516)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 16, 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-GFP
FEATURES             Location/Qualifiers
     source          1..9516
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     intron          32..259
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      271..280
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             280..345
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             352..372
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(397..420,421..423,424..4497)
                     /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             397..4497
                     /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             4498..4545
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4561..4614
                     /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             join(4615..4617,4618..5328)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4615..4617,4618..5328)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4918..4937)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5265..5286
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5356..5373)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5362..5569
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5578..5718
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5578..5707
                     /label=AAV2 ITR
     rep_origin      5793..6248
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5880..5899)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6090..6111
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6265..6284)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6384..6406
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6444..6462)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6530..6634
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6635..6703,6704..7495)
                     /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(6853..6872)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7666..8254
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8155..8174
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8316..8564
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        8592..8667
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        8792..9040
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        9067..9142
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(9151..9169)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(9152..9169)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        9206..9491
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt
       61 gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc
      121 acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagctga gcaagaggta
      181 agggtttaag ggatggttgg ttggtggggt attaatgttt aattacctgg agcacctgcc
      241 tgaaatcact ttttttcagg ttggaccggt gccaccatgg actataagga ccacgacgga
      301 gactacaagg atcatgatat tgattacaaa gacgatgacg ataagatggc cccaaagaag
      361 aagcggaagg tcggtatcca cggagtccca gcagccgaca agaagtacag catcggcctg
      421 gccatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc
      481 aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga
      541 gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga
      601 agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag
      661 atggccaagg tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag
      721 gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac
      781 gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc
      841 gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg
      901 atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg
      961 cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag
     1021 gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg
     1081 cccggcgaga agaagaatgg cctgttcggc aacctgattg ccctgagcct gggcctgacc
     1141 cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac
     1201 acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg
     1261 tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac
     1321 accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac
     1381 caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag
     1441 attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag
     1501 gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg
     1561 ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc
     1621 atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt
     1681 tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc
     1741 tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc
     1801 gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag
     1861 agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc
     1921 aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac
     1981 gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg
     2041 gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc
     2101 aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc
     2161 tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat
     2221 gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga
     2281 gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag
     2341 cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc
     2401 atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc
     2461 aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag
     2521 aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc
     2581 agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa
     2641 gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc
     2701 acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa
     2761 gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag
     2821 aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac
     2881 atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac
     2941 gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac
     3001 gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc
     3061 aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc
     3121 gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag
     3181 cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg
     3241 atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat
     3301 ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg
     3361 aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg
     3421 tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc
     3481 ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag
     3541 attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc
     3601 ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg
     3661 ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct
     3721 atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag
     3781 aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg
     3841 gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg
     3901 gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa
     3961 gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc
     4021 cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc
     4081 tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc
     4141 gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc
     4201 atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa
     4261 gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc
     4321 atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc
     4381 accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac
     4441 cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacaaa
     4501 aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaaggaatt cggcagtgga
     4561 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccagtgagc
     4621 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     4681 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     4741 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     4801 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     4861 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     4921 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     4981 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     5041 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     5101 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     5161 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     5221 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     5281 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga attctaacta
     5341 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     5401 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     5461 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     5521 aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg
     5581 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     5641 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     5701 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     5761 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     5821 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     5881 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     5941 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     6001 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     6061 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     6121 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     6181 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     6241 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     6301 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     6361 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     6421 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     6481 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     6541 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     6601 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     6661 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     6721 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     6781 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     6841 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     6901 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     6961 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     7021 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     7081 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     7141 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     7201 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     7261 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     7321 tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc
     7381 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     7441 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     7501 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     7561 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     7621 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     7681 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     7741 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     7801 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     7861 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     7921 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     7981 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     8041 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     8101 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     8161 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     8221 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     8281 gttcctggcc ttttgctggc cttttgctca catgtgaggg cctatttccc atgattcctt
     8341 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     8401 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     8461 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     8521 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggggtc ttcggatcca
     8581 tgcgaagacc tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     8641 cttgaaaaag tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa
     8701 aataaggcta gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg ctctagatta
     8761 accctcacta aagggaaagg tcgggcagga agagggccta tttcccatga ttccttcata
     8821 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
     8881 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     8941 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     9001 tttcttggct ttatatatct tgtggaaagg acgaaacacc gggagaccat cgatgagagg
     9061 gtctcagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     9121 aaaagtggca ccgagtcggt gctttttttt tctatagtgt cacctaaatt gcagctctgg
     9181 cccgtgtctc aaaatctctg gtacccgtta cataacttac ggtaaatggc ccgcctggct
     9241 gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg actttccatt
     9301 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     9361 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattgtg
     9421 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     9481 ctattaccat ggtcgaggtg agccccacgt tctgct
//
