LOCUS       sequence-275335-        8451 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            SpCas9 with a c-terminal bi-partite NLS, 3x flag tag, and P2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8451)
  AUTHORS   Walton RT, Christie KA, Whittaker MN, Kleinstiver BP
  TITLE     Unconstrained genome targeting with near-PAMless engineered 
            CRISPR-Cas9 variants.
  JOURNAL   Science. 2020 Apr 17;368(6488):290-296. doi: 
            10.1126/science.aba8853. Epub 2020 Mar 26.
  PUBMED    32217751
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: 
            "10.1126/science.aba8853"; journalName: "Science"; date: 
            "2020-04-17- 17"; volume: "368"; issue: "6488"; pages: "290-296"
COMMENT     Sequence Label: pCMV-T7-SpCas9-P2A-EGFP (RTW3027)
FEATURES             Location/Qualifiers
     source          1..8451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             3992..4012
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4025..4090
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4109..4165
                     /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(4166..4168,4169..4171,4172..4885)
                     /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(4166..4168,4169..4171,4172..4882)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4472..4491)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4819..4840
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4891..4908
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4931..4948)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4937..5161
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5232..5248)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5232..5248)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5245..5267)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5256..5272
                     /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(5280..5286,5287..5304,5305..5310))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5325..5346
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5463..5480)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5634..6222)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5714..5733)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6393..7184,7185..7253))
                     /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     7016..7035
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7254..7358)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7437..7456)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7628..8007
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8008..8211
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8162..8182
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     8253..8272
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8253..8271
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      8279..8288
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(8285..8451,1..3937)
                     /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
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(8285..8451,1..3937)
                     /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
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa
       61 gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag
      121 cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca
      181 ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta
      241 ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct
      301 ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc
      361 cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt
      421 cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact
      481 gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg
      541 cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt
      601 cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga
      661 caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct
      721 gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc
      781 cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa
      841 agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa
      901 gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat
      961 caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga
     1021 ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct
     1081 gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa
     1141 ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc
     1201 caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg
     1261 gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac
     1321 caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga
     1381 gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa
     1441 gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa
     1501 ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga
     1561 ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga
     1621 tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct
     1681 ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct
     1741 gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata
     1801 caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg
     1861 caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct
     1921 gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca
     1981 gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg
     2041 catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc
     2101 cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa
     2161 cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct
     2221 gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct
     2281 gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta
     2341 cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt
     2401 gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt
     2461 gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa
     2521 gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt
     2581 catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga
     2641 ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat
     2701 caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg
     2761 cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc
     2821 cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta
     2881 cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta
     2941 cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga
     3001 gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa
     3061 gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa
     3121 aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag
     3181 cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag
     3241 ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa
     3301 actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa
     3361 gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat
     3421 caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc
     3481 tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct
     3541 gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca
     3601 gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt
     3661 ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa
     3721 gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac
     3781 caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta
     3841 caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta
     3901 cgagacacgg atcgacctgt ctcagctggg aggcgacgga tccggcggag gcggaagcgg
     3961 gaaaagaacc gccgacggca gcgaattcga gcccaagaag aagaggaaag tctcgagcgg
     4021 aggcgactac aaagaccatg acggtgatta taaagatcat gacatcgatt acaaggatga
     4081 cgatgacaag ggaggcggtg gaagcggcgc aacaaacttc tctctgctga aacaagccgg
     4141 agatgtcgaa gagaatcctg gaccgatggt gagcaagggc gaggagctgt tcaccggggt
     4201 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     4261 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     4321 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     4381 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     4441 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     4501 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     4561 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     4621 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     4681 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     4741 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     4801 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     4861 cggcatggac gagctgtaca agtgaccggt catcatcacc atcaccattg agtttaaacc
     4921 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     4981 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     5041 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     5101 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     5161 gcttctgagg cggaaagaac cagctggggc tcgataccgt cgacctctag ctagagcttg
     5221 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5281 aacatacgag ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc
     5341 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5401 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5461 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5521 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5581 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5641 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5701 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5761 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5821 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5881 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5941 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6001 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6061 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6121 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6181 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6241 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6301 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6361 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6421 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6481 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6541 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6601 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6661 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6721 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6781 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6841 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6901 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6961 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7021 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7081 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7141 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7201 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7261 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7321 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7381 cctgacgtcg acggatcggg agatcgatct cccgatcccc tagggtcgac tctcagtaca
     7441 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
     7501 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
     7561 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
     7621 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
     7681 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     7741 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     7801 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     7861 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     7921 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     7981 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
     8041 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
     8101 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
     8161 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg
     8221 aaccgtcaga tccgctagag atccgcggcc gctaatacga ctcactatag ggagagccgc
     8281 caccatggac aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc
     8341 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga
     8401 ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg g
//
