LOCUS       sequence-275521-        8451 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            SpCas9-VQR(D1135V/R1335Q/T1337R) 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-VQR-P2A-EGFP (RTW3520)
FEATURES             Location/Qualifiers
     source          1..8451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        131..510
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        511..714
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     665..685
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     756..775
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        756..774
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      782..791
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(788..4189,4190..4192,4193..4789,4790..4792,4793..4795,
                     4796..4798,4799..4891)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             788..4891
                     /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
                     ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4946..4966
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4979..5044
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5063..5119
                     /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(5120..5122,5123..5125,5126..5839)
                     /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(5120..5122,5123..5125,5126..5836)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5426..5445)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5773..5794
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5845..5862
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5885..5902)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5891..6115
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6186..6202)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6186..6202)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6199..6221)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6210..6226
                     /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(6234..6240,6241..6258,6259..6264))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6279..6300
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6417..6434)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6588..7176)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6668..6687)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7347..8138,8139..8207))
                     /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     7970..7989
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8208..8312)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8391..8410)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
       61 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
      121 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      181 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      241 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      301 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      361 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      421 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      481 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      541 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      601 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      661 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta
      721 gtgaaccgtc agatccgcta gagatccgcg gccgctaata cgactcacta tagggagagc
      781 cgccaccatg gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg
      841 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac
      901 cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac
      961 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg
     1021 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt
     1081 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat
     1141 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct
     1201 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
     1261 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa
     1321 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga
     1381 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa
     1441 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt
     1501 cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
     1561 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct
     1621 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga
     1681 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag
     1741 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct
     1801 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
     1861 ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc
     1921 catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct
     1981 gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga
     2041 gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga
     2101 aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg
     2161 aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt
     2221 cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt
     2281 cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt
     2341 caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc
     2401 cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa
     2461 agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt
     2521 ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct
     2581 gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga
     2641 tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac
     2701 ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg
     2761 ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac
     2821 aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca
     2881 cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga
     2941 tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct
     3001 gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa
     3061 catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg
     3121 cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga
     3181 acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa
     3241 tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt
     3301 ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac
     3361 cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa
     3421 gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga
     3481 caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa
     3541 gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg
     3601 gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct
     3661 gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat
     3721 caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat
     3781 caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt
     3841 gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt
     3901 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
     3961 gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg
     4021 ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac
     4081 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa
     4141 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg tgagccccac
     4201 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
     4261 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
     4321 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
     4381 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg
     4441 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
     4501 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
     4561 tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa
     4621 gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg
     4681 ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
     4741 gggagcccct gccgccttca agtactttga caccaccatc gaccggaagc agtacagaag
     4801 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
     4861 acggatcgac ctgtctcagc tgggaggcga cggatccggc ggaggcggaa gcgggaaaag
     4921 aaccgccgac ggcagcgaat tcgagcccaa gaagaagagg aaagtctcga gcggaggcga
     4981 ctacaaagac catgacggtg attataaaga tcatgacatc gattacaagg atgacgatga
     5041 caagggaggc ggtggaagcg gcgcaacaaa cttctctctg ctgaaacaag ccggagatgt
     5101 cgaagagaat cctggaccga tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     5161 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     5221 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     5281 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     5341 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     5401 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     5461 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     5521 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     5581 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     5641 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     5701 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     5761 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     5821 ggacgagctg tacaagtgac cggtcatcat caccatcacc attgagttta aacccgctga
     5881 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     5941 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     6001 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     6061 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     6121 gaggcggaaa gaaccagctg gggctcgata ccgtcgacct ctagctagag cttggcgtaa
     6181 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     6241 cgagccggaa gcataaagtg taaagcctag ggtgcctaat gagtgagcta actcacatta
     6301 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     6361 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     6421 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     6481 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     6541 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6601 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6661 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     6721 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6781 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6841 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6901 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6961 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     7021 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     7081 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     7141 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     7201 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     7261 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     7321 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     7381 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     7441 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     7501 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7561 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7621 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7681 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7741 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7801 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7861 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7921 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7981 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     8041 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     8101 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     8161 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     8221 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     8281 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     8341 gtcgacggat cgggagatcg atctcccgat cccctagggt cgactctcag tacaatctgc
     8401 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga g
//
