LOCUS       sequence-197599-        8697 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses Cas9 nuclease and gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8697)
  AUTHORS   Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T
  TITLE     Multiplex genome engineering in human cells using all-in-one 
            CRISPR/Cas9 vector system.
  JOURNAL   Sci Rep. 2014 Jun 23;4:5400. doi: 10.1038/srep05400.
  PUBMED    24954249
REFERENCE   2  (bases 1 to 8697)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2014-06-23"; volume: "4:5400. doi"; pages: " 
            10.1038/srep05400"
COMMENT     Sequence Label: pX330S-2
FEATURES             Location/Qualifiers
     source          1..8697
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..293
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        295..572
                     /label=chicken beta-actin promoter
     intron          573..800
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      812..821
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             821..886
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             893..913
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             938..5038
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             938..5038
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5039..5086
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5114..5131)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5120..5327
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5336..5476
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5336..5465
                     /label=AAV2 ITR
     rep_origin      5551..6006
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5638..5657)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5848..5869
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6023..6042)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6142..6164
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6202..6220)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             6627..7418
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      7601..8189
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8090..8109
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8267..8515
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8267..8287
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8438..8457
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8534..8609
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tgagacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
       61 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      121 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      181 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat
      241 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
      301 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
      361 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg
      421 cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg
      481 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
      541 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg
      601 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
      661 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt
      721 aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc
      781 ctgaaatcac tttttttcag gttggaccgg tgccaccatg gactataagg accacgacgg
      841 agactacaag gatcatgata ttgattacaa agacgatgac gataagatgg ccccaaagaa
      901 gaagcggaag gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct
      961 ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag
     1021 caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg
     1081 agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag
     1141 aagaagatac accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga
     1201 gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga
     1261 ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca
     1321 cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc
     1381 cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct
     1441 gatcgagggc gacctgaacc ccgacaacag cgacgttgac aagctgttca tccagctggt
     1501 gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa
     1561 ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct
     1621 gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac
     1681 ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga
     1741 cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct
     1801 gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa
     1861 caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca
     1921 ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga
     1981 gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca
     2041 ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact
     2101 gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag
     2161 catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt
     2221 ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc
     2281 ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag
     2341 cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca
     2401 gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc
     2461 caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata
     2521 cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt
     2581 ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt
     2641 caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc
     2701 ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa
     2761 tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag
     2821 agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa
     2881 gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg
     2941 catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc
     3001 caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca
     3061 gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg
     3121 cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa
     3181 agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac
     3241 cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa
     3301 agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga
     3361 gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga
     3421 catcaaccgg ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga
     3481 cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa
     3541 cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc
     3601 caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag
     3661 cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa
     3721 gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct
     3781 gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga
     3841 tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct
     3901 gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt
     3961 gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat
     4021 cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga
     4081 gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac
     4141 cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat
     4201 gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc
     4261 tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa
     4321 gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt
     4381 ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat
     4441 ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga
     4501 agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg
     4561 ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc
     4621 ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc
     4681 cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat
     4741 catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa
     4801 agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat
     4861 catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac
     4921 caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca
     4981 ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa
     5041 aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagtaag aattcctaga
     5101 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     5161 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     5221 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     5281 gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg gccgcaggaa
     5341 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     5401 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     5461 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5521 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     5581 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     5641 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     5701 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5761 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5821 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5881 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     5941 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     6001 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     6061 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     6121 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     6181 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     6241 taataatggt ttcttagacg tcaggtggca cttccagcca ggacagaaat gcctcgactt
     6301 cgctgctacc caaggttgcc gggtgacgca caccgtggaa acggatgaag gcacgaaccc
     6361 agtggacata agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca
     6421 actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg
     6481 cttgttatga ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc
     6541 gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca
     6601 gtcgccctaa aacaaagtta aacattatga gggaagcggt gatcgccgaa gtatcgactc
     6661 aactatcaga ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac
     6721 atttgtacgg ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg
     6781 ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg
     6841 aaacttcggc ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg
     6901 tgcacgacga catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat
     6961 ggcagcgcaa tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg
     7021 ctatcttgct gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg
     7081 aactctttga tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc
     7141 tatggaactc gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc
     7201 gcatttggta cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg
     7261 caatggagcg cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc
     7321 ttggacaaga agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact
     7381 acgtgaaagg cgagatcacc aaggtagtcg gcaaataaaa gcattggtaa ctgtcagacc
     7441 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     7501 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     7561 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     7621 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     7681 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     7741 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     7801 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     7861 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     7921 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     7981 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     8041 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     8101 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     8161 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     8221 tggccttttg ctggcctttt gctcaggtct cactctagag gcatgtgagg gcctatttcc
     8281 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     8341 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     8401 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     8461 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgggtc
     8521 ttcgagaaga cctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     8581 aacttgaaaa agtggcaccg agtcggtgct tttttgtttt agagctagaa atagcaagtt
     8641 aaaataaggc tagtccgttt ttagcgcgtg cgccaattct gcagacaaat ggctcta
//
