LOCUS       sequence-454380-        9737 bp    DNA     circular SYN 15-DEC-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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9737)
  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 9737)
  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-GFP
FEATURES             Location/Qualifiers
     source          1..9737
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(3201..3221)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3228..3293)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      3293..3302
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          3314..3541
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     promoter        complement(3542..3817)
                     /label=chicken beta-actin promoter
     enhancer        3819..4104
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        4141..4159
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4141..4158
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        complement(4168..4243)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(4270..4518)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        complement(4643..4718)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(4746..4994)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     rep_origin      complement(5056..5644)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5136..5155)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5815..6606,6607..6675))
                     /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     6438..6457
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6676..6780)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6848..6866
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6904..6926)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7026..7045
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(7062..7517)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7199..7220)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7411..7430
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   7592..7732
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7603..7732
                     /label=AAV2 ITR
     polyA_signal    7741..7948
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     7937..7954
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(7982..8692,8693..8695))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(7982..8692,8693..8695))
                     /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(8024..8045)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     8373..8392
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             complement(8696..8749)
                     /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             complement(8765..8812)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(join(8813..9737,1..3149,3150..3152,3153..3176))
                     /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             complement(join(8813..9737,1..3176))
                     /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"
ORIGIN
        1 cgcttccgga tctcgccgtt ggccagggta atctcggtct tgaaaaagtt catgatgttg
       61 ctgtagaaga agtacttggc ggtagccttg ccgatttcct gctcgctctt ggcgatcatc
      121 ttccgcacgt cgtacacctt gtagtcgccg tacacgaact cgctttccag cttagggtac
      181 tttttgatca gggcggttcc cacgacggcg ttcaggtagg cgtcgtgggc gtggtggtag
      241 ttgttgatct cgcgcacttt gtaaaactgg aaatccttcc ggaaatcgga caccagcttg
      301 gacttcaggg tgatcacttt cacttcccgg atcagcttgt cattctcgtc gtacttagtg
      361 ttcatccggg agtccaggat ctgtgccacg tgctttgtga tctgccgggt ttccaccagc
      421 tgtctcttga tgaagccggc cttatccagt tcgctcaggc cgcctctctc ggccttggtc
      481 agattgtcga actttctctg ggtaatcagc ttggcgttca gcagctgccg ccagtagttc
      541 ttcatcttct tcacgacctc ttcggagggc acgttgtcgc tcttgccccg gttcttgtcg
      601 cttctggtca gcaccttgtt gtcgatggag tcgtccttca gaaagctctg aggcacgata
      661 tggtccacat cgtagtcgga cagccggttg atgtccagtt cctggtccac gtacatatcc
      721 cgcccattct gcaggtagta caggtacagc ttctcgttct gcagctgggt gttttccacg
      781 gggtgttctt tcaggatctg gctgcccagc tctttgatgc cctcttcgat ccgcttcatt
      841 ctctcgcggc tgttcttctg tcccttctgg gtggtctggt tctctctggc catttcgatc
      901 acgatgttct cgggcttgtg ccggcccatc actttcacga gctcgtccac caccttcact
      961 gtctgcagga tgcccttctt aatggcgggg ctgccggcca gattggcaat gtgctcgtgc
     1021 aggctatcgc cctggccgga cacctgggct ttctggatgt cctctttaaa ggtcaggctg
     1081 tcgtcgtgga tcagctgcat gaagtttctg ttggcgaagc cgtcggactt caggaaatcc
     1141 aggattgtct tgccggactg cttgtcccgg atgccgttga tcagcttccg gctcagcctg
     1201 ccccagccgg tgtatctccg ccgcttcagc tgcttcatca ctttgtcgtc gaacaggtgg
     1261 gcataggttt tcagccgttc ctcgatcatc tctctgtcct caaacagtgt cagggtcagc
     1321 acgatatctt ccagaatgtc ctcgttttcc tcattgtcca ggaagtcctt gtccttgata
     1381 attttcagca gatcgtggta tgtgcccagg gaggcgttga accgatcttc cacgccggag
     1441 atttccacgg agtcgaagca ctcgattttc ttgaagtagt cctctttcag ctgcttcacg
     1501 gtcactttcc ggttggtctt gaacagcagg tccacgatgg cctttttctg ctcgccgctc
     1561 aggaaggcgg gctttctcat tccctcggtc acgtatttca ctttggtcag ctcgttatac
     1621 acggtgaagt actcgtacag caggctgtgc ttgggcagca ccttctcgtt gggcaggttc
     1681 ttatcgaagt tggtcatccg ctcgatgaag ctctgggcgg aagcgccctt gtccaccact
     1741 tcctcgaagt tccagggggt gatggtttcc tcgctctttc tggtcatcca ggcgaatctg
     1801 ctgtttcccc tggccagagg gcccacgtag taggggatgc ggaaggtcag gatcttctcg
     1861 atcttttccc ggttgtcctt caggaatggg taaaaatctt cctgccgccg cagaatggcg
     1921 tgcagctctc ccaggtggat ctggtggggg atgctgccgt tgtcgaaggt ccgctgcttc
     1981 cgcagcaggt cctctctgtt cagcttcacg agcagttcct cggtgccgtc catcttttcc
     2041 aggatgggct tgatgaactt gtagaactct tcctggctgg ctccgccgtc aatgtagccg
     2101 gcgtagccgt tcttgctctg gtcgaagaaa atctctttgt acttctcagg cagctgctgc
     2161 cgcacgagag ctttcagcag ggtcaggtcc tggtggtgct cgtcgtatct cttgatcata
     2221 gaggcgctca ggggggcctt ggtgatctcg gtgttcactc tcaggatgtc gctcagcagg
     2281 atggcgtcgg acaggttctt ggcggccaga aacaggtcgg cgtactggtc gccgatctgg
     2341 gccagcaggt tgtccaggtc gtcgtcgtag gtgtccttgc tcagctgcag tttggcatcc
     2401 tcggccaggt cgaagttgct cttgaagttg ggggtcaggc ccaggctcag ggcaatcagg
     2461 ttgccgaaca ggccattctt cttctcgccg ggcagctggg cgatcagatt ttccagccgt
     2521 ctgctcttgc tcagtctggc agacaggatg gccttggcgt ccacgccgct ggcgttgatg
     2581 gggttttcct cgaacagctg gttgtaggtc tgcaccagct ggatgaacag cttgtccacg
     2641 tcgctgttgt cggggttcag gtcgccctcg atcaggaagt ggccccggaa cttgatcatg
     2701 tgggccaggg ccagatagat cagccgcagg tcggccttgt cggtgctgtc caccagtttc
     2761 tttctcaggt ggtagatggt ggggtacttc tcgtggtagg ccacctcgtc cacgatgttg
     2821 ccgaagatgg ggtgccgctc gtgcttctta tcctcttcca ccaggaagga ctcttccagt
     2881 ctgtggaaga agctgtcgtc caccttggcc atctcgttgc tgaagatctc ttgcagatag
     2941 cagatccggt tcttccgtct ggtgtatctt cttctggcgg ttctcttcag ccgggtggcc
     3001 tcggctgttt cgccgctgtc gaacagcagg gctccgatca ggttcttctt gatgctgtgc
     3061 cggtcggtgt tgcccagcac cttgaatttc ttgctgggca ccttgtactc gtcggtgatc
     3121 acggcccagc ccacagagtt ggtgccgatg gccaggccga tgctgtactt cttgtcggct
     3181 gctgggactc cgtggatacc gaccttccgc ttcttctttg gggccatctt atcgtcatcg
     3241 tctttgtaat caatatcatg atccttgtag tctccgtcgt ggtccttata gtccatggtg
     3301 gcaccggtcc aacctgaaaa aaagtgattt caggcaggtg ctccaggtaa ttaaacatta
     3361 ataccccacc aaccaaccat cccttaaacc cttacctctt gctcagctaa ttacagcccg
     3421 gaggagaagg gccgtcccgc ccgctcacct gtgggagtaa cgcggtcagt cagagccggg
     3481 gcgggcggcg cgaggcggcg gcggagcggg gcacggggcg aaggcagcgc gcagcgactc
     3541 ccgcccgccg cgcgcttcgc tttttatagg gccgccgccg ccgccgcctc gccataaaag
     3601 gaaactttcg gagcgcgccg ctctgattgg ctgccgccgc acctctccgc ctcgccccgc
     3661 cccgcccctc gccccgcccc gccccgcccg gcgcgcgccc cccccccccc cgcccccacc
     3721 gctgcacaaa ataattaaaa aataaataaa tacaaaattg ggggtgggga ggggggggag
     3781 atggggagag tgaagcagaa cgtggggctc acctcgacca tggtaatagc gatgactaat
     3841 acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc acaatgccag
     3901 gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga
     3961 tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc
     4021 cctattggcg ttactattga cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg
     4081 ccatttaccg taagttatgt aacgggtacc agagattttg agacacgggc cagagctgca
     4141 atttaggtga cactatagaa aaaaaaagca ccgactcggt gccacttttt caagttgata
     4201 acggactagc cttattttaa cttgctattt ctagctctaa aactgagacc ctctcatcga
     4261 tggtctcccg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt
     4321 tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta
     4381 cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt
     4441 caaattaatt ctaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat
     4501 catgggaaat aggccctctt cctgcccgac ctttcccttt agtgagggtt aatctagagc
     4561 catttgtctg cagaattggc gcacgcgcta aaaacggact agccttattt taacttgcta
     4621 tttctagctc taaaacaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga
     4681 ctagccttat tttaacttgc tatttctagc tctaaaacag gtcttcgcat ggatccgaag
     4741 accccggtgt ttcgtccttt ccacaagata tataaagcca agaaatcgaa atactttcaa
     4801 gttacggtaa gcatatgata gtccatttta aaacataatt ttaaaactgc aaactaccca
     4861 agaaattatt actttctacg tcacgtattt tgtactaata tctttgtgtt tacagtcaaa
     4921 ttaattccaa ttatctctct aacagccttg tatcgtatat gcaaatatga aggaatcatg
     4981 ggaaataggc cctcacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5041 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5101 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5161 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5221 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5281 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5341 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5401 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5461 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5521 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5581 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5641 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5701 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5761 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5821 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5881 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5941 tgcaatgata ccgcggcttc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6001 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6061 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6121 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6181 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6241 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6301 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6361 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6421 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6481 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6541 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6601 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6661 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6721 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6781 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     6841 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     6901 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     6961 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     7021 ctatgcggca tcagagcaga ttgtactgag agtgcaccat aaaattgtaa acgttaatat
     7081 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aatagaccga
     7141 aatcggcaaa atcccttata aatcaaaaga atagcccgag atagagttga gtgttgttcc
     7201 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     7261 cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt ttttggggtc
     7321 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     7381 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctaa
     7441 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     7501 gccgctacag ggcgcgtact atggttgctt tgacgtatgc ggtgtgaaat accgcacaga
     7561 tgcgtaagga gaaaataccg catcaggcgc ccctgcaggc agctgcgcgc tcgctcgctc
     7621 actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg
     7681 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc
     7741 tccccagcat gcctgctatt ctcttcccaa tcctccccct tgctgtcctg ccccacccca
     7801 ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag
     7861 gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca
     7921 acagatggct ggcaactaga aggcacagtc gaggctgatc agcgagctct agttagaatt
     7981 ccttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga
     8041 ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt
     8101 ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg
     8161 cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt
     8221 tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt
     8281 gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca
     8341 gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga
     8401 agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct
     8461 tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg
     8521 tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt
     8581 aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt
     8641 ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcactgggc
     8701 caggattctc ctcgacgtca ccgcatgtta gcagacttcc tctgccctct ccactgccga
     8761 attccttttt cttttttgcc tggccggcct ttttcgtggc cgccggcctt ttgtcgcctc
     8821 ccagctgaga caggtcgatc cgtgtctcgt acaggccggt gatgctctgg tggatcaggg
     8881 tggcgtccag cacctctttg gtgctggtgt acctcttccg gtcgatggtg gtgtcaaagt
     8941 acttgaaggc ggcaggggct cccagattgg tcagggtaaa caggtggatg atattctcgg
     9001 cctgctctct gatgggctta tcccggtgct tgttgtaggc ggacagcact ttgtccagat
     9061 tagcgtcggc caggatcact ctcttggaga actcgctgat ctgctcgatg atctcgtcca
     9121 ggtagtgctt gtgctgttcc acaaacagct gtttctgctc attatcctcg ggggagccct
     9181 tcagcttctc atagtggctg gccaggtaca ggaagttcac atatttggag ggcagggcca
     9241 gttcgtttcc cttctgcagt tcgccggcag aggccagcat tctcttccgg ccgttttcca
     9301 gctcgaacag ggagtactta ggcagcttga tgatcaggtc ctttttcact tctttgtagc
     9361 ccttggcttc cagaaagtcg atgggattct tctcgaagct gcttctttcc atgatggtga
     9421 tccccagcag ctctttcaca ctcttcagtt tcttggactt gcccttttcc actttggcca
     9481 ccaccagcac agaataggcc acggtggggc tgtcgaagcc gccgtacttc ttagggtccc
     9541 agtccttctt tctggcgatc agcttatcgc tgttcctctt gggcaggata gactctttgc
     9601 tgaagccgcc tgtctgcacc tcggtctttt tcacgatatt cacttggggc atgctcagca
     9661 ctttccgcac ggtggcaaaa tcccggccct tatcccacac gatctccccg gtttcgccgt
     9721 ttgtctcgat cagaggc
//
