LOCUS       sequence-267863-        9010 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FRB fused SpCas9 for NanoMEDIC packaging..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9010)
  AUTHORS   Gee P, Lung MSY, Okuzaki Y, Sasakawa N, Iguchi T, Makita Y, Hozumi 
            H, Miura Y, Yang LF, Iwasaki M, Wang XH, Waller MA, Shirai N, Abe 
            YO, Fujita Y, Watanabe K, Kagita A, Iwabuchi KA, Yasuda M, Xu H, 
            Noda T, Komano J, Sakurai H, Inukai N, Hotta A
  TITLE     Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and 
            sgRNA to induce therapeutic exon skipping.
  JOURNAL   Nat Commun. 2020 Mar 13;11(1):1334. doi: 10.1038/s41467-020-14957-y.
  PUBMED    32170079
REFERENCE   2  (bases 1 to 9010)
  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"; journalName: "Nat 
            Commun."; date: "2020-03-13"; volume: "11(1):1334. doi"; pages: " 
            10.1038/s41467-020-14957-y"
COMMENT     Sequence Label: pHLS-EF1a-FRB-SpCas9-A
FEATURES             Location/Qualifiers
     source          1..9010
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      166..301
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     418..436
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        546..1728
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          777..1719
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1676..1696
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1750..1774
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1783..1792
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1801..2079
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             2086..6189
                     /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
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2086..6189
                     /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
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     RBS             6102..6110
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             6196..6243
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6250..6330
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             6346..6366
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(6379..6403)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(6568..6587)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6633..6688
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6687..6706)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(7104..7121)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7275..7863)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7355..7374)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8034..8825,8826..8894))
                     /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     8657..8676
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8895..8999)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaaagtgcc acctgacgtc gctagcgttt aaacccgggc gatcgctcta gattgtctca
       61 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
      121 ttccccgaaa agtgccacct gggactagct ttttgcaaaa gcctaggcct ccaaaaaagc
      181 ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa
      241 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
      301 tgggcggagt taggggcggg actatggttg ctgactaatt gagatgagct tgcatgccga
      361 cattgattat tgactagtcc ctaagaaacc attcttatca tgacattaac ctataaaaat
      421 aggcgtatca cgaggcctag actagtgcgg ccgctgcaga agctttgcaa agatggataa
      481 agttttaaac agagaggaat ctttgcagct aatggacctt ctaggtcttg aaaggagtgg
      541 gaattggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
      601 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg
      661 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
      721 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa
      781 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt
      841 gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga
      901 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
      961 gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     1021 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     1081 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt
     1141 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg
     1201 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     1261 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     1321 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     1381 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     1441 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     1501 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt
     1561 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     1621 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     1681 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaga attagcggtg
     1741 gcggccatca caagtttgta caaaaaagca ggctgtcgag ccgccaccat ggcttctaga
     1801 atcctctggc atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg
     1861 gaaaggaacg tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg
     1921 ggcccccaga ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag
     1981 gcccaagagt ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cctccaagcc
     2041 tgggacctct attatcatgt gttccgacga atctcaaagt cgaccatgga taagaaatac
     2101 agcattggac tggacattgg gacaaactcc gtgggatggg ccgtgattac agacgaatac
     2161 aaagtgcctt caaagaagtt caaggtgctg ggcaacaccg atagacacag catcaagaaa
     2221 aatctgattg gagccctgct gttcgactcc ggcgagacag ctgaagcaac tcggctgaaa
     2281 agaactgctc ggagaaggta tacccgccga aagaatagga tctgctacct gcaggagatt
     2341 ttcagcaacg aaatggccaa ggtggacgat agtttctttc accgcctgga ggaatcattc
     2401 ctggtcgagg aagataagaa acacgagcgg catcccatct ttggcaacat tgtggacgag
     2461 gtcgcttatc acgaaaagta ccctaccatc tatcatctga ggaagaaact ggtggactcc
     2521 acagataaag cagacctgcg cctgatctat ctggccctgg ctcacatgat taagttccgg
     2581 ggccattttc tgatcgaggg ggatctgaac ccagacaatt ctgatgtgga caagctgttc
     2641 atccagctgg tccagacata caatcagctg tttgaggaaa accccattaa tgcatctggc
     2701 gtggacgcaa aagccatcct gagtgccaga ctgtctaaga gtcggagact ggagaacctg
     2761 atcgctcagc tgccagggga aaagaaaaac ggcctgtttg ggaatctgat tgcactgtca
     2821 ctgggactga ctcccaactt caagagcaat tttgatctgg ccgaggacgc taaactgcag
     2881 ctgtccaagg acacctatga cgatgacctg gataacctgc tggctcagat cggggatcag
     2941 tacgcagacc tgttcctggc cgctaagaat ctgtctgacg ccatcctgct gagtgatatt
     3001 ctgcgcgtga acaccgagat tacaaaagcc cccctgtcag ctagcatgat caagagatat
     3061 gacgagcacc atcaggatct gaccctgctg aaggctctgg tgaggcagca gctgcctgag
     3121 aagtacaagg aaatcttctt tgatcagtct aagaacggat acgccggcta tattgacggc
     3181 ggggctagtc aggaggagtt ctacaagttt atcaaaccca ttctggagaa gatggatggc
     3241 acagaggaac tgctggtgaa actgaatcgg gaagacctgc tgaggaagca gcgcactttt
     3301 gataacggaa gcatccctca ccagattcat ctgggagagc tgcacgcaat cctgaggcgc
     3361 caggaagact tctacccatt tctgaaggat aacagggaga agatcgaaaa aattctgaca
     3421 ttccgcatcc cctactatgt gggccctctg gcaagaggca acagccggtt tgcctggatg
     3481 actcgcaaat ctgaggaaac aatcactccc tggaacttcg aggaagtggt cgataagggc
     3541 gcttccgcac agtctttcat tgagcggatg acaaacttcg acaagaacct gccaaacgaa
     3601 aaagtgctgc ccaagcactc tctgctgtac gagtatttca cagtctataa cgaactgact
     3661 aaggtgaaat acgtcaccga ggggatgaga aagcctgcct tcctgagtgg agaacagaag
     3721 aaagctatcg tggacctgct gtttaaaacc aataggaagg tgacagtcaa gcagctgaaa
     3781 gaggactatt tcaagaaaat tgaatgtttc gattctgtgg agatcagtgg cgtcgaagac
     3841 aggtttaacg cctccctggg gacctaccac gatctgctga agatcattaa ggataaagac
     3901 ttcctggaca acgaggaaaa tgaggatatc ctggaagaca ttgtgctgac cctgacactg
     3961 tttgaggata gggaaatgat cgaggaacgc ctgaagacct atgcccatct gttcgatgac
     4021 aaagtgatga aacagctgaa gcgacggaga tacacaggat ggggccgact gtctcggaag
     4081 ctgatcaatg ggattcgcga caaacagagt ggaaagacca tcctggactt tctgaaatca
     4141 gatggcttcg ccaaccggaa cttcatgcag ctgattcacg atgacagcct gacattcaaa
     4201 gaggatatcc agaaggcaca ggtgtccggg cagggagact ctctgcacga gcatatcgca
     4261 aacctggccg gcagccctgc catcaagaaa gggattctgc agaccgtgaa ggtggtggac
     4321 gagctggtga aagtcatggg aagacataag ccagaaaaca tcgtgattga gatggccagg
     4381 gaaaatcaga ccacacagaa aggccagaag aactcaaggg agcgcatgaa aagaatcgag
     4441 gaaggaatta aggaactggg cagccagatc ctgaaagagc accccgtgga aaacacacag
     4501 ctgcagaatg agaagctgta tctgtactat ctgcagaatg gacgcgatat gtacgtggac
     4561 caggagctgg atattaaccg actgtccgat tacgacgtgg atcatatcgt cccacagtca
     4621 ttcctgaaag atgacagcat tgacaataag gtgctgaccc gctctgacaa aaaccgaggc
     4681 aagagtgata atgtcccctc agaggaagtg gtcaagaaaa tgaagaacta ctggaggcag
     4741 ctgctgaatg ccaaactgat cacacagcga aagtttgata acctgactaa agctgagcgg
     4801 ggaggcctga gtgaactgga caaagcaggc ttcattaagc gacagctggt ggagacacgg
     4861 cagatcacaa agcacgtcgc ccagattctg gattcaagaa tgaacactaa gtacgatgag
     4921 aatgacaaac tgatcagaga agtgaaggtc attaccctga agtcaaaact ggtgagcgac
     4981 tttcggaaag atttccagtt ttataaggtc agagagatca acaactacca ccatgctcat
     5041 gacgcatacc tgaacgcagt ggtcggcaca gccctgatta agaaataccc taaactggag
     5101 tccgagttcg tgtacgggga ctataaggtg tacgatgtca gaaaaatgat cgccaagtct
     5161 gagcaggaaa ttggcaaagc cactgctaag tatttctttt acagtaacat catgaatttc
     5221 tttaagactg agatcaccct ggcaaatggg gaaatccgaa agcggccact gattgagact
     5281 aacggcgaga caggagaaat cgtgtgggac aaaggaagag attttgctac cgtgaggaag
     5341 gtcctgagca tgccccaagt gaatattgtc aagaaaacag aggtgcagac tgggggattc
     5401 agtaaggaat caattctgcc taaacgcaac tccgataagc tgatcgcccg aaagaaagac
     5461 tgggacccca agaagtatgg cgggttcgac tccccaactg tggcttactc tgtcctggtg
     5521 gtcgcaaagg tggagaaggg aaaaagcaag aaactgaaat ccgtcaagga actgctgggc
     5581 atcaccatta tggagcgcag ctccttcgaa aagaatccta tcgattttct ggaggccaaa
     5641 ggctataagg aagtgaagaa agacctgatc atcaagctgc caaagtactc actgtttgag
     5701 ctggaaaacg ggagaaagag gatgctggca agcgccgggg agctgcagaa aggaaatgaa
     5761 ctggccctgc cctccaagta cgtgaacttc ctgtatctgg ctagccacta cgagaagctg
     5821 aaagggtccc ctgaggataa cgaacagaaa cagctgtttg tggagcagca caagcattat
     5881 ctggacgaga tcattgaaca gattagcgag ttctccaaaa gagtgatcct ggctgacgca
     5941 aatctggata aggtcctgag cgcatacaac aaacaccggg ataagccaat cagagagcag
     6001 gccgaaaata tcattcatct gttcactctg accaacctgg gagcccccgc agccttcaag
     6061 tattttgaca ctaccatcga tcgcaaacga tacacaagca ctaaggaggt gctggacgct
     6121 accctgattc atcagagcat tactggcctg tatgaaacaa ggattgacct gtctcagctg
     6181 ggcggcgact ccggaaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa
     6241 aagggatcct acccatacga tgttccagat tacgcttacc catacgatgt tccagattac
     6301 gcttacccat acgatgttcc agattacgct taatccggag ctgaccccaa gaagaagagg
     6361 aaggtgtgag acctctagac ccagctttct tgtacaaagt ggtgatctag ctaaaacgcg
     6421 gcctcgaatc gaattcgatt cgaatgtaca gatctggatc ctaggcttaa gtatacctcg
     6481 agttaaattc actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa
     6541 tgccctggct cacaaatacc actgagatct ttttccctct gccaaaaatt atggggacat
     6601 catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat
     6661 agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt
     6721 aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata tgctggctgc
     6781 catgaacaaa ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca
     6841 ttccttattc catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt
     6901 gttatttttt tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt
     6961 cctcctctcc tgactactcc cagtcatagc tgtccctctt ctcttatgga gatccctcga
     7021 cttaattaaa ccggtcagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     7081 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     7141 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     7201 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     7261 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     7321 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     7381 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     7441 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     7501 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     7561 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     7621 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     7681 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     7741 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     7801 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     7861 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     7921 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     7981 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     8041 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     8101 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     8161 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     8221 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     8281 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     8341 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     8401 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     8461 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     8521 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     8581 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     8641 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     8701 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     8761 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     8821 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     8881 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     8941 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     9001 acatttcccc
//
