LOCUS       Exported                8954 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses Cas9 nickase and gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    pX330A_D10A-1x7
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8954)
  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 8954)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8954
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        8..293
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          569..796
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      808..817
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             817..882
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             889..909
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             934..5034
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             934..5034
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5035..5082
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5110..5127)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5116..5323
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5332..5472
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5332..5461
                     /label=AAV2 ITR
     rep_origin      5547..6002
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5634..5653)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5844..5865
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6019..6038)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6138..6160
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6198..6216)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6284..6388
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6389..7249
                     /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     complement(6607..6626)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7420..8008
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7909..7928
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8070..8310
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     8070..8090
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8241..8260
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8337..8412
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(8498..8803)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL"
     primer_bind     8653..8675
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8667..8684
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8668..8684
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8694..8713
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8694..8712
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8760..8780)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(8760..8778)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8799..8815)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8799..8815)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8812..8834)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8823..8839
                     /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(8847..8877)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8892..8913
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 aggtacccgt 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 ggcgcgcgcc
      421 aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg cggcggcagc
      481 caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc
      541 ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc
      601 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca
      661 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca agaggtaagg
      721 gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga
      781 aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca cgacggagac
      841 tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc aaagaagaag
      901 cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat cggcctggcc
      961 atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag
     1021 aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc
     1081 ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga
     1141 agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg
     1201 gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat
     1261 aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag
     1321 aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac
     1381 ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc
     1441 gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag
     1501 acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc
     1561 atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc
     1621 ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc
     1681 aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc
     1741 tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt
     1801 ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc
     1861 gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag
     1921 gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt
     1981 ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa
     2041 gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc
     2101 gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc
     2161 ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac
     2221 ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac
     2281 tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag
     2341 gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc
     2401 ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag
     2461 cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg
     2521 accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac
     2581 ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag
     2641 aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc
     2701 ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag
     2761 gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag
     2821 atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag
     2881 ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc
     2941 cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac
     3001 agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa
     3061 gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc
     3121 cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg
     3181 atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc
     3241 cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag
     3301 ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag
     3361 ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc
     3421 aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac
     3481 tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg
     3541 ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag
     3601 ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa
     3661 ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac
     3721 gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc
     3781 cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc
     3841 cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac
     3901 gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac
     3961 ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc
     4021 aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt
     4081 accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg
     4141 gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc
     4201 caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc
     4261 ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag
     4321 tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa
     4381 aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa
     4441 agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg
     4501 aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg
     4561 aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc
     4621 aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag
     4681 gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc
     4741 gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg
     4801 ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc
     4861 cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc
     4921 atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag
     4981 agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaaaagg
     5041 ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agtaagaatt cctagagctc
     5101 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     5161 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     5221 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     5281 gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg caggaacccc
     5341 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     5401 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     5461 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     5521 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     5581 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     5641 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     5701 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     5761 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     5821 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     5881 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     5941 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     6001 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     6061 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     6121 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     6181 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     6241 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     6301 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     6361 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     6421 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     6481 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     6541 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     6601 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     6661 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     6721 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6781 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6841 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6901 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6961 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     7021 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     7081 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg
     7141 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     7201 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     7261 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     7321 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     7381 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     7441 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     7501 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     7561 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     7621 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     7681 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7741 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7801 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     7861 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     7921 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7981 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     8041 ggccttttgc tggccttttg ctcacatgtg agggcctatt tcccatgatt ccttcatatt
     8101 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     8161 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     8221 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     8281 tcttggcttt atatatcttg tggaaaggac gaaacaccgg gtcttcgaga agacctgttt
     8341 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     8401 ccgagtcggt gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     8461 tttttagcgc gtgcgccaat tctgcagaca aatggctcta gagaccggcg ccgctacagg
     8521 gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     8581 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     8641 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg
     8701 actcactata gggcgaattg ggtaccgggc cccccctcga ggtcctccag cttttgttcc
     8761 ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     8821 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     8881 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     8941 caccggtggt ctct
//
