LOCUS       sequence-363405-        8451 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV and T7 promoter expression plasmid for human codon usage 
            nSpCas9(D10A) 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(D10A)-BPNLS(SV40)-3xFLAG-P2A-E...
FEATURES             Location/Qualifiers
     source          1..8451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        183..386
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     337..357
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     428..447
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        428..446
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      454..463
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(460..486,487..489,490..4563)
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             460..4563
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4618..4638
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4651..4716
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4735..4791
                     /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(4792..4794,4795..4797,4798..5511)
                     /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(4792..4794,4795..4797,4798..5508)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5098..5117)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5445..5466
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5517..5534
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5557..5574)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5563..5787
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5858..5874)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5858..5874)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5871..5893)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5882..5898
                     /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(5906..5912,5913..5930,5931..5936))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5951..5972
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6089..6106)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6260..6848)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6340..6359)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7019..7810,7811..7879))
                     /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     7642..7661
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7880..7984)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8063..8082)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8254..8451,1..182)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
       61 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      121 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      181 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      241 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      301 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      361 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagagatccg
      421 cggccgctaa tacgactcac tatagggaga gccgccacca tggacaagaa gtacagcatc
      481 ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
      541 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
      601 atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc
      661 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
      721 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
      781 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
      841 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
      901 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
      961 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
     1021 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
     1081 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
     1141 cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc
     1201 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
     1261 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     1321 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
     1381 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
     1441 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     1501 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc
     1561 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     1621 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
     1681 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
     1741 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
     1801 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
     1861 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc
     1921 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
     1981 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg
     2041 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc
     2101 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
     2161 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
     2221 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
     2281 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
     2341 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
     2401 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
     2461 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
     2521 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
     2581 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
     2641 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
     2701 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
     2761 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
     2821 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
     2881 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
     2941 ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg
     3001 aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc
     3061 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg
     3121 aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc
     3181 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
     3241 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac
     3301 aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
     3361 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
     3421 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
     3481 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
     3541 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
     3601 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
     3661 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg
     3721 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
     3781 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac
     3841 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
     3901 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
     3961 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
     4021 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
     4081 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
     4141 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
     4201 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac
     4261 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
     4321 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag
     4381 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
     4441 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
     4501 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
     4561 gacggatccg gcggaggcgg aagcgggaaa agaaccgccg acggcagcga attcgagccc
     4621 aagaagaaga ggaaagtctc gagcggaggc gactacaaag accatgacgg tgattataaa
     4681 gatcatgaca tcgattacaa ggatgacgat gacaagggag gcggtggaag cggcgcaaca
     4741 aacttctctc tgctgaaaca agccggagat gtcgaagaga atcctggacc gatggtgagc
     4801 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     4861 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     4921 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     4981 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     5041 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     5101 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     5161 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     5221 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     5281 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     5341 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     5401 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     5461 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagtg accggtcatc
     5521 atcaccatca ccattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     5581 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     5641 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     5701 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     5761 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga
     5821 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5881 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5941 agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     6001 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     6061 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     6121 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     6181 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     6241 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     6301 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6361 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6421 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6481 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6541 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6601 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6661 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6721 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6781 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6841 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6901 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6961 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     7021 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     7081 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     7141 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     7201 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     7261 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     7321 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7381 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7441 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7501 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7561 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7621 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7681 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7741 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7801 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7861 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7921 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7981 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg
     8041 atcccctagg gtcgactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     8101 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     8161 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     8221 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat
     8281 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     8341 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     8401 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa t
//
