LOCUS       sequence-362676-        8451 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV and T7 promoter expression plasmid for human codon usage 
            nSpCas9(H840A) with a C-terminal bi-partite NLS, 3x FLAG, and 
            P2A-eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8451)
  AUTHORS   Alonso-Lerma B, Jabalera Y, Samperio S, Morin M, Fernandez A, Hille 
            LT, Silverstein RA, Quesada-Ganuza A, Reifs A, Fernandez-Penalver S,
            Benitez Y, Soletto L, Gavira JA, Diaz A, Vranken W, Sanchez-Mejias 
            A, Guell M, Mojica FJM, Kleinstiver BP, Moreno-Pelayo MA, Montoliu 
            L, Perez-Jimenez R
  TITLE     Evolution of CRISPR-associated endonucleases as inferred from 
            resurrected proteins.
  JOURNAL   Nat Microbiol. 2023 Jan;8(1):77-90. doi: 10.1038/s41564-022-01265-y.
            Epub 2023 Jan 2.
  PUBMED    36593295
REFERENCE   2  (bases 1 to 8451)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41564-022-01265-y"; journalName: "Nat Microbiol"; date: 
            "2023-01"; volume: "8"; issue: "1"; pages: "77-90"
COMMENT     Sequence Label: pCMV-T7-nSpCas9(H840A)-BPNLS(SV40)-3xFLAG-P2A-...
FEATURES             Location/Qualifiers
     source          1..8451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        188..391
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     342..362
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     433..452
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        433..451
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      459..468
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(465..2981,2982..2984,2985..4568)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(H840A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the H840A mutation in the HNH catalytic domain"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             465..4568
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4623..4643
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4656..4721
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4740..4796
                     /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             join(4797..4799,4800..4802,4803..5516)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4797..4799,4800..4802,4803..5513)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5103..5122)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5450..5471
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5522..5539
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5562..5579)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5568..5792
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5863..5879)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5863..5879)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5876..5898)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5887..5903
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5911..5917,5918..5935,5936..5941))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5956..5977
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6094..6111)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6265..6853)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6345..6364)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7024..7815,7816..7884))
                     /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     7647..7666
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7885..7989)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8068..8087)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8259..8451,1..187)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
       61 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      121 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      181 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
      241 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
      301 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
      361 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagag
      421 atccgcggcc gctaatacga ctcactatag ggagagccgc caccatggac aagaagtaca
      481 gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca
      541 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga
      601 acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga
      661 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct
      721 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc
      781 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg
      841 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca
      901 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg
      961 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca
     1021 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg
     1081 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga
     1141 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc
     1201 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc
     1261 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt
     1321 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc
     1381 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg
     1441 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga
     1501 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg
     1561 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca
     1621 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg
     1681 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc
     1741 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct
     1801 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga
     1861 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg
     1921 cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga
     1981 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca
     2041 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa
     2101 aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag
     2161 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc
     2221 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact
     2281 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt
     2341 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca
     2401 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc
     2461 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg
     2521 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag
     2581 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca
     2641 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg
     2701 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag
     2761 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag
     2821 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc
     2881 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc
     2941 aggaactgga catcaaccgg ctgtccgact acgatgtgga cgccatcgtg cctcagagct
     3001 ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca
     3061 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc
     3121 tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag
     3181 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc
     3241 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga
     3301 atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt
     3361 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg
     3421 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa
     3481 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg
     3541 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt
     3601 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa
     3661 acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag
     3721 tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca
     3781 gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact
     3841 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg
     3901 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga
     3961 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg
     4021 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc
     4081 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac
     4141 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga
     4201 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc
     4261 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta
     4321 atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg
     4381 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt
     4441 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca
     4501 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg
     4561 gaggcgacgg atccggcgga ggcggaagcg ggaaaagaac cgccgacggc agcgaattcg
     4621 agcccaagaa gaagaggaaa gtctcgagcg gaggcgacta caaagaccat gacggtgatt
     4681 ataaagatca tgacatcgat tacaaggatg acgatgacaa gggaggcggt ggaagcggcg
     4741 caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgatgg
     4801 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     4861 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     4921 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     4981 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     5041 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     5101 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     5161 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     5221 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     5281 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     5341 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     5401 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     5461 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtgaccgg
     5521 tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg tgccttctag
     5581 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     5641 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     5701 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     5761 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     5821 ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     5881 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5941 agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     6001 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     6061 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     6121 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     6181 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     6241 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     6301 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6361 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6421 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6481 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6541 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6601 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6661 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6721 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6781 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6841 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6901 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6961 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     7021 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     7081 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     7141 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     7201 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7261 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     7321 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     7381 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7441 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7501 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7561 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7621 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7681 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7741 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7801 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7861 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7921 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7981 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatcgatc
     8041 tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat agttaagcca
     8101 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     8161 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     8221 tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta
     8281 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     8341 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     8401 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac g
//
