LOCUS       sequence-142912-        9271 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 VRER variant that detects NGCG PAM, combined with 2A_GFP for 
            expression control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9271)
  AUTHORS   Paquet D, Kwart D, Chen A, Sproul A, Jacob S, Teo S, Olsen KM, Gregg
            A, Noggle S, Tessier-Lavigne M
  TITLE     Efficient introduction of specific homozygous and heterozygous 
            mutations using CRISPR/Cas9.
  JOURNAL   Nature. 2016 May 5;533(7601):125-9. doi: 10.1038/nature17664. Epub 
            2016 Apr 27.
  PUBMED    27120160
REFERENCE   2  (bases 1 to 9271)
  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/nature17664"; journalName: "Nature"; date: "2016-05-5- 5"; 
            volume: "533"; issue: "7601"; pages: "125-9"
COMMENT     Sequence Label: pCas9-VRER_2A_GFP
FEATURES             Location/Qualifiers
     source          1..9271
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3402,3403..3405,3406..3651,3652..3654,3655..4002,
                     4003..4005,4006..4008,4009..4011,4012..4104)
                     /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             1..4104
                     /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             4117..4137
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4153..4206
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(4207..4209,4210..4212,4213..4926)
                     /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(4207..4209,4210..4212,4213..4923)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4252..4273)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4513..4532)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4860..4881
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4969..5090
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5006..5025)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5060..5079
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5139..5155)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5139..5155)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5152..5174)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5163..5179
                     /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(5187..5193,5194..5211,5212..5217))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5232..5253
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5370..5387)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5541..6129)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5621..5640)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6300..7091,7092..7160))
                     /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     6923..6942
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7161..7265)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7344..7363)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7535..7914
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7917..8192
                     /label=chicken beta-actin promoter
     intron          8193..9205
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     9129..9151
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     9213..9232
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      join(9266..9271,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc
       61 attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc
      121 cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa
      181 gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc
      241 tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg
      301 ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc
      361 aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag
      421 aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat
      481 atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat
      541 gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg
      601 atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg
      661 cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat
      721 cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa
      781 gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc
      841 cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt
      901 ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt
      961 atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga
     1021 cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc
     1081 ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg
     1141 gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc
     1201 aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac
     1261 gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt
     1321 gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc
     1381 agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa
     1441 gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa
     1501 aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt
     1561 tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg
     1621 tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc
     1681 gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc
     1741 agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc
     1801 attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc
     1861 ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct
     1921 catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg
     1981 cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg
     2041 gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac
     2101 tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt
     2161 cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc
     2221 gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt
     2281 atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg
     2341 atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca
     2401 gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg
     2461 gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat
     2521 atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc
     2581 gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa
     2641 aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg
     2701 actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag
     2761 cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac
     2821 accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct
     2881 aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat
     2941 taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa
     3001 tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa
     3061 atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc
     3121 aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga
     3181 ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc
     3241 gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta
     3301 cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc
     3361 gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgtttctcc tacagtcgct
     3421 tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc
     3481 aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac
     3541 tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag
     3601 tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gcgcgagctg
     3661 cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc
     3721 cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa
     3781 caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg
     3841 atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag
     3901 cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg
     3961 cctgcagcct tcaagtactt cgacaccacc atagacagaa aggagtaccg ctctacaaag
     4021 gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc
     4081 gacctctctc agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgagg
     4141 tccggcggcg gagagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc
     4201 ggcccaatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     4261 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     4321 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     4381 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     4441 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     4501 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     4561 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     4621 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     4681 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     4741 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     4801 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     4861 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     4921 aagtaactgc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     4981 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     5041 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     5101 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     5161 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     5221 tagggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     5281 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5341 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5401 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5461 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5521 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5581 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5641 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5701 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5761 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5821 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5881 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5941 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6001 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6061 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6121 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6181 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6241 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6301 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6361 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6421 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6481 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6541 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6601 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6661 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6721 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6781 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6841 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6901 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6961 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7021 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7081 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7141 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7201 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7261 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tcgatctccc
     7321 gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     7381 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
     7441 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
     7501 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa
     7561 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     7621 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     7681 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac
     7741 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     7801 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     7861 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catgggtcga
     7921 ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt
     7981 gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggcgcg
     8041 cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg
     8101 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
     8161 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgttgcct tcgccccgtg
     8221 ccccgctccg cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac
     8281 aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac
     8341 ggctcgtttc ttttctgtgg ctgcgtgaaa gccttaaagg gctccgggag ggccctttgt
     8401 gcggggggga gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc
     8461 ggcccgcgct gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc
     8521 gcgtgtgcgc gaggggagcg cggccggggg cggtgccccg cggtgcgggg gggctgcgag
     8581 gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg
     8641 gcggtcgggc tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc
     8701 ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg
     8761 gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga
     8821 ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg
     8881 ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga
     8941 gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg
     9001 cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct
     9061 tctccatctc cagcctcggg gctgtccgca gggggacggc tgccttcggg ggggacgggg
     9121 cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agtgcctctg ctaaccatgt
     9181 tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca
     9241 tcattttggc aaagaattcg cggccgccac c
//
