LOCUS       sequence-302011-        8481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutisite Gateway mediated vector construction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8481)
  AUTHORS   Yoshimatsu S, Sone T, Nakajima M, Sato T, Okochi R, Ishikawa M, 
            Nakamura M, Sasaki E, Shiozawa S, Okano H
  TITLE     A versatile toolbox for knock-in gene targeting based on the 
            Multisite Gateway technology.
  JOURNAL   PLoS One. 2019 Aug 27;14(8):e0221164. doi: 
            10.1371/journal.pone.0221164. eCollection 2019.
  PUBMED    31454364
REFERENCE   2  (bases 1 to 8481)
  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: "PLoS One.";
            date: "2019-08-27"; volume: "14(8):e0221164. doi"; pages: " 
            10.1371/journal.pone.0221164. eCollection 2019"
COMMENT     Sequence Label: pENTR-L3-Csy4-FokI-dCas9-EGFP-L2
FEATURES             Location/Qualifiers
     source          1..8481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     159..178
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(393..420)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(512..598)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    652..747
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      776..785
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1346..1399
                     /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             1403..1423
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1430..2017
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     CDS             join(2033..5431,5432..5434,5435..6031,6032,6033..6035,
                     6036..6037,6038..6040,6041..6133)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(2033..2056,2057..2059,2060..4546,4547..4549,
                     4550..6133)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6140..6160
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6188..6253
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             join(6260..6262,6263..6265,6266..6979)
                     /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(6260..6262,6263..6265,6266..6976)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6305..6326)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6566..6585)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6913..6934
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(6977..7076)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7188..7207)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             7199..8008
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(7266..7285)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(8151..8481,1..258)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
       61 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
      121 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
      181 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
      241 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
      301 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
      361 accgctagcc aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg atggccttct
      421 gcttagtttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt
      481 gcttcacaac gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg
      541 acaaacaaca gataaaacga aaggcccagt cttccgactg agcctttcgt tttatttgat
      601 gcctggcagt tccctactct cgcgttaacg ctagcatgga tctcgggccc caaataatga
      661 ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa tgctttttta
      721 taatgccaac tttgtataat aaagttgggt accgcgggcc cgggatccac cggtcgccac
      781 catgggtgat cattatctgg atattcggct gaggcctgat ccagagttcc cacctgcgca
      841 gctgatgtct gtcctttttg gcaaacttca tcaggccctg gttgcccagg gcggagatcg
      901 gataggggta agctttccag acctcgacga aagccggagc cgcctgggag aacgcctgcg
      961 gatccacgct tctgccgacg atctgagagc cttgctggca aggccatggc ttgaggggct
     1021 ccgggatcac ctgcagtttg gcgaacccgc cgttgttccc cacccaaccc cttatcggca
     1081 ggtgtctaga gtgcaggcca aatctaatcc agaacggctg cgacggcgac tcatgcggcg
     1141 acatgatctt agcgaggaag aggcccgaaa aagaatccct gataccgtgg cccgcgccct
     1201 tgacttgcct tttgtcacac tgcggtccca gagtacgggg cagcatttca gacttttcat
     1261 tcgacacggg ccactgcaag ttaccgccga agaaggaggc tttacttgtt atggactctc
     1321 caagggaggt ttcgtgccct ggtttgaggg cagaggaagt ctgttaacat gcggtgacgt
     1381 cgaggagaat cctggcccaa tgcctaagaa gaagcggaag gtgagcagcc aacttgtgaa
     1441 gtctgaactc gaggagaaaa aatcagagtt gagacacaag ttgaagtacg tgccacacga
     1501 atacatcgag cttatcgaga tcgccagaaa cagtacccag gataggatcc ttgagatgaa
     1561 agtcatggag ttctttatga aggtctacgg ttatagagga aagcaccttg gcggtagcag
     1621 aaagcccgat ggcgccatct atactgtcgg atctcctatc gattatgggg tgatcgtgga
     1681 taccaaagct tactcaggcg ggtacaactt gcccatagga caagccgacg agatgcagcg
     1741 gtatgtcgaa gagaaccaga cgcgcaacaa gcacatcaac cccaatgaat ggtggaaagt
     1801 gtacccaagt agtgtgactg agttcaagtt cctgtttgtc tccggccact ttaagggcaa
     1861 ttataaagct cagctcacta gactcaatca catcacaaac tgcaacggag ctgtgttgtc
     1921 agtggaggag ctcctgattg gaggcgagat gatcaaagcc ggcaccctta cactggagga
     1981 ggtgcggcgg aagttcaaca atggagagat caacttcggt ggcggtggat ccgataaaaa
     2041 gtattctatt ggtttagcca tcggcactaa ttccgttgga tgggctgtca taaccgatga
     2101 atacaaagta ccttcaaaga aatttaaggt gttggggaac acagaccgtc attcgattaa
     2161 aaagaatctt atcggtgccc tcctattcga tagtggcgaa acggcagagg cgactcgcct
     2221 gaaacgaacc gctcggagaa ggtatacacg tcgcaagaac cgaatatgtt acttacaaga
     2281 aatttttagc aatgagatgg ccaaagttga cgattctttc tttcaccgtt tggaagagtc
     2341 cttccttgtc gaagaggaca agaaacatga acggcacccc atctttggaa acatagtaga
     2401 tgaggtggca tatcatgaaa agtacccaac gatttatcac ctcagaaaaa agctagttga
     2461 ctcaactgat aaagcggacc tgaggttaat ctacttggct cttgcccata tgataaagtt
     2521 ccgtgggcac tttctcattg agggtgatct aaatccggac aactcggatg tcgacaaact
     2581 gttcatccag ttagtacaaa cctataatca gttgtttgaa gagaacccta taaatgcaag
     2641 tggcgtggat gcgaaggcta ttcttagcgc ccgcctctct aaatcccgac ggctagaaaa
     2701 cctgatcgca caattacccg gagagaagaa aaatgggttg ttcggtaacc ttatagcgct
     2761 ctcactaggc ctgacaccaa attttaagtc gaacttcgac ttagctgaag atgccaaatt
     2821 gcagcttagt aaggacacgt acgatgacga tctcgacaat ctactggcac aaattggaga
     2881 tcagtatgcg gacttatttt tggctgccaa aaaccttagc gatgcaatcc tcctatctga
     2941 catactgaga gttaatactg agattaccaa ggcgccgtta tccgcttcaa tgatcaaaag
     3001 gtacgatgaa catcaccaag acttgacact tctcaaggcc ctagtccgtc agcaactgcc
     3061 tgagaaatat aaggaaatat tctttgatca gtcgaaaaac gggtacgcag gttatattga
     3121 cggcggagcg agtcaagagg aattctacaa gtttatcaaa cccatattag agaagatgga
     3181 tgggacggaa gagttgcttg taaaactcaa tcgcgaagat ctactgcgaa agcagcggac
     3241 tttcgacaac ggtagcattc cacatcaaat ccacttaggc gaattgcatg ctatacttag
     3301 aaggcaggag gatttttatc cgttcctcaa agacaatcgt gaaaagattg agaaaatcct
     3361 aacctttcgc ataccttact atgtgggacc cctggcccga gggaactctc ggttcgcatg
     3421 gatgacaaga aagtccgaag aaacgattac tccatggaat tttgaggaag ttgtcgataa
     3481 aggtgcgtca gctcaatcgt tcatcgagag gatgaccaac tttgacaaga atttaccgaa
     3541 cgaaaaagta ttgcctaagc acagtttact ttacgagtat ttcacagtgt acaatgaact
     3601 cacgaaagtt aagtatgtca ctgagggcat gcgtaaaccc gcctttctaa gcggagaaca
     3661 gaagaaagca atagtagatc tgttattcaa gaccaaccgc aaagtgacag ttaagcaatt
     3721 gaaagaggac tactttaaga aaattgaatg cttcgattct gtcgagatct ccggggtaga
     3781 agatcgattt aatgcgtcac ttggtacgta tcatgacctc ctaaagataa ttaaagataa
     3841 ggacttcctg gataacgaag agaatgaaga tatcttagaa gatatagtgt tgactcttac
     3901 cctctttgaa gatcgggaaa tgattgagga aagactaaaa acatacgctc acctgttcga
     3961 cgataaggtt atgaaacagt taaagaggcg tcgctatacg ggctggggac gattgtcgcg
     4021 gaaacttatc aacgggataa gagacaagca aagtggtaaa actattctcg attttctaaa
     4081 gagcgacggc ttcgccaata ggaactttat gcagctgatc catgatgact ctttaacctt
     4141 caaagaggat atacaaaagg cacaggtttc cggacaaggg gactcattgc acgaacatat
     4201 tgcgaatctt gctggttcgc cagccatcaa aaagggcata ctccagacag tcaaagtagt
     4261 ggatgagcta gttaaggtca tgggacgtca caaaccggaa aacattgtaa tcgagatggc
     4321 acgcgaaaat caaacgactc agaaggggca aaaaaacagt cgagagcgga tgaagagaat
     4381 agaagagggt attaaagaac tgggcagcca gatcttaaag gagcatcctg tggaaaatac
     4441 ccaattgcag aacgagaaac tttacctcta ttacctacaa aatggaaggg acatgtatgt
     4501 tgatcaggaa ctggacataa accgtttatc tgattacgac gtcgatgcca ttgtacccca
     4561 atcctttttg aaggacgatt caatcgacaa taaagtgctt acacgctcgg ataagaaccg
     4621 agggaaaagt gacaatgttc caagcgagga agtcgtaaag aaaatgaaga actattggcg
     4681 gcagctccta aatgcgaaac tgataacgca aagaaagttc gataacttaa ctaaagctga
     4741 gaggggtggc ttgtctgaac ttgacaaggc cggatttatt aaacgtcagc tcgtggaaac
     4801 ccgccaaatc acaaagcatg ttgcacagat actagattcc cgaatgaata cgaaatacga
     4861 cgagaacgat aagctgattc gggaagtcaa agtaatcact ttaaagtcaa aattggtgtc
     4921 ggacttcaga aaggattttc aattctataa agttagggag ataaataact accaccatgc
     4981 gcacgacgct tatcttaatg ccgtcgtagg gaccgcactc attaagaaat acccgaagct
     5041 agaaagtgag tttgtgtatg gtgattacaa agtttatgac gtccgtaaga tgatcgcgaa
     5101 aagcgaacag gagataggca aggctacagc caaatacttc ttttattcta acattatgaa
     5161 tttctttaag acggaaatca ctctggcaaa cggagagata cgcaaacgac ctttaattga
     5221 aaccaatggg gagacaggtg aaatcgtatg ggataagggc cgggacttcg cgacggtgag
     5281 aaaagttttg tccatgcccc aagtcaacat agtaaagaaa actgaggtgc agaccggagg
     5341 gttttcaaag gaatcgattc ttccaaaaag gaatagtgat aagctcatcg ctcgtaaaaa
     5401 ggactgggac ccgaaaaagt acggtggctt cgatagccct acagttgcct attctgtcct
     5461 agtagtggca aaagttgaga agggaaaatc caagaaactg aagtcagtca aagaattatt
     5521 ggggataacg attatggagc gctcgtcttt tgaaaagaac cccatcgact tccttgaggc
     5581 gaaaggttac aaggaagtaa aaaaggatct cataattaaa ctaccaaagt atagtctgtt
     5641 tgagttagaa aatggccgaa aacggatgtt ggctagcgcc ggagagcttc aaaaggggaa
     5701 cgaactcgca ctaccgtcta aatacgtgaa tttcctgtat ttagcgtccc attacgagaa
     5761 gttgaaaggt tcacctgaag ataacgaaca gaagcaactt tttgttgagc agcacaaaca
     5821 ttatctcgac gaaatcatag agcaaatttc ggaattcagt aagagagtca tcctagctga
     5881 tgccaatctg gacaaagtat taagcgcata caacaagcac agggataaac ccatacgtga
     5941 gcaggcggaa aatattatcc atttgtttac tcttaccaac ctcggcgctc cagccgcatt
     6001 caagtatttt gacacaacga tagatcgcaa acgatacact tctaccaagg aggtgctaga
     6061 cgcgacactg attcaccaat ccatcacggg attatatgaa actcggatag atttgtcaca
     6121 gcttgggggt gacggatccc ccaagaagaa gaggaaagtc tcgagtagcc gcgctaagcg
     6181 ggcaccggtg aaacagactt tgaattttga ccttctcaag ttggcgggag acgtggagtc
     6241 caacccaggg ccggatatca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     6301 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     6361 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     6421 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     6481 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     6541 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     6601 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     6661 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     6721 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     6781 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     6841 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     6901 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     6961 ggacgagctg tacaagtaac agctttcttg tacaaagttg gcattataag aaagcattgc
     7021 ttatcaattt gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat
     7081 ccagctgcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa
     7141 tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     7201 gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt
     7261 atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt
     7321 gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa
     7381 tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac
     7441 catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg
     7501 aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc
     7561 gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag
     7621 cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc
     7681 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca
     7741 taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa
     7801 ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc
     7861 agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt
     7921 acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt
     7981 tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca
     8041 gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag
     8101 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     8161 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     8221 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     8281 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     8341 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     8401 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     8461 tcgggctgaa cggggggttc g
//
