LOCUS       sequence-275523-        8451 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            SpCas9-VRER(D1135V/G1218R/R1335E/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-VRER-P2A-EGFP (RTW3160)
FEATURES             Location/Qualifiers
     source          1..8451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        161..540
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        541..744
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     695..715
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     786..805
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        786..804
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      812..821
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(818..4219,4220..4222,4223..4468,4469..4471,4472..4819,
                     4820..4822,4823..4825,4826..4828,4829..4921)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/G1218R/R1335E/T1337R mutations"
                     /label=Cas9 VRER
                     /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
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             818..4921
                     /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
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4976..4996
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5009..5074
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5093..5149
                     /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(5150..5152,5153..5155,5156..5869)
                     /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(5150..5152,5153..5155,5156..5866)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5456..5475)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5803..5824
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5875..5892
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5915..5932)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5921..6145
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6216..6232)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6216..6232)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6229..6251)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6240..6256
                     /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(6264..6270,6271..6288,6289..6294))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6309..6330
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6447..6464)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6618..7206)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6698..6717)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7377..8168,8169..8237))
                     /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     8000..8019
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8238..8342)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8421..8440)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
       61 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
      121 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
      181 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      241 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      301 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      361 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      421 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      481 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      541 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      601 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      661 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      721 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gagatccgcg
      781 gccgctaata cgactcacta tagggagagc cgccaccatg gacaagaagt acagcatcgg
      841 cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
      901 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
      961 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
     1021 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
     1081 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
     1141 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
     1201 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
     1261 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
     1321 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
     1381 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
     1441 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
     1501 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct
     1561 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
     1621 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
     1681 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
     1741 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
     1801 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
     1861 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag
     1921 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
     1981 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
     2041 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
     2101 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
     2161 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
     2221 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
     2281 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
     2341 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
     2401 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
     2461 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
     2521 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
     2581 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
     2641 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
     2701 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
     2761 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
     2821 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
     2881 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
     2941 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
     3001 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
     3061 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
     3121 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
     3181 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
     3241 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
     3301 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa
     3361 ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga
     3421 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa
     3481 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct
     3541 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
     3601 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa
     3661 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
     3721 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
     3781 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt
     3841 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
     3901 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
     3961 cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga
     4021 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag
     4081 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
     4141 gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc
     4201 taagaagtac ggcggcttcg tgagccccac cgtggcctat tctgtgctgg tggtggccaa
     4261 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
     4321 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
     4381 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
     4441 cggccggaag agaatgctgg cctctgccag agaactgcag aagggaaacg aactggccct
     4501 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
     4561 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga
     4621 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
     4681 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa
     4741 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga
     4801 caccaccatc gaccggaagg agtacagaag caccaaagag gtgctggacg ccaccctgat
     4861 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga
     4921 cggatccggc ggaggcggaa gcgggaaaag aaccgccgac ggcagcgaat tcgagcccaa
     4981 gaagaagagg aaagtctcga gcggaggcga ctacaaagac catgacggtg attataaaga
     5041 tcatgacatc gattacaagg atgacgatga caagggaggc ggtggaagcg gcgcaacaaa
     5101 cttctctctg ctgaaacaag ccggagatgt cgaagagaat cctggaccga tggtgagcaa
     5161 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     5221 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     5281 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     5341 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     5401 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     5461 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     5521 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     5581 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     5641 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     5701 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     5761 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     5821 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtgac cggtcatcat
     5881 caccatcacc attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     5941 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     6001 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     6061 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     6121 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgata
     6181 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     6241 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag
     6301 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     6361 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     6421 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     6481 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     6541 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     6601 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     6661 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     6721 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     6781 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     6841 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     6901 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6961 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     7021 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     7081 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     7141 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     7201 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     7261 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     7321 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     7381 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     7441 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     7501 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     7561 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     7621 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     7681 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     7741 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     7801 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     7861 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     7921 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7981 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     8041 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     8101 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     8161 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     8221 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     8281 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     8341 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat
     8401 cccctagggt cgactctcag tacaatctgc tctgatgccg catagttaag c
//
