LOCUS       sequence-305039-        7848 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 7848)
  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 7848)
  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-Cas9-EGFP-L2
FEATURES             Location/Qualifiers
     source          1..7848
                     /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             782..1342
                     /codon_start=1
                     /product="CRISPR-associated endoribonuclease responsible 
                     for pre-crRNA processing in Pseudomonas aeruginosa 
                     (Haurwitz et al., 2010)"
                     /label=Csy4
                     /translation="MDHYLDIRLRPDPEFPPAQLMSVLFGKLHQALVAQGGDRIGVSFP
                     DLDESRSRLGERLRIHASADDLRALLARPWLEGLRDHLQFGEPAVVPHPTPYRQVSRVQ
                     AKSNPERLRRRLMRRHDLSEEEARKRIPDTVARALDLPFVTLRSQSTGQHFRLFIRHGP
                     LQVTAEEGGFTCYGLSKGGFVPWF"
     CDS             1343..1396
                     /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(1397..4798,4799..4801,4802..5398,5399,5400..5402,
                     5403..5404,5405..5407,5408..5500)
                     /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="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             1397..5500
                     /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             1397..5500
                     /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             5507..5527
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5555..5620
                     /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(5627..5629,5630..5632,5633..6346)
                     /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(5627..5629,5630..5632,5633..6343)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5672..5693)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5933..5952)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6280..6301
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(6344..6443)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6555..6574)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6566..7375
                     /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(6633..6652)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(7518..7848,1..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 catggaccac tacctcgaca ttcgcttgcg accggacccg gaatttcccc cggcgcaact
      841 catgagcgtg ctcttcggca agctccacca ggccctggtg gcacagggcg gggacaggat
      901 cggcgtgagc ttccccgacc tcgacgaaag ccgctcccgg ctgggcgagc gcctgcgcat
      961 tcatgcctcg gcggacgacc ttcgtgccct gctcgcccgg ccctggctgg aagggttgcg
     1021 ggaccatctg caattcggag aaccggcagt cgtgcctcac cccacaccgt accgtcaggt
     1081 cagtcgggtt caggcgaaaa gcaatccgga acgcctgcgg cggcggctca tgcgccggca
     1141 cgatctgagt gaggaggagg ctcggaaacg cattcccgat acggtcgcga gagccttgga
     1201 cctgcccttc gtcacgctac gcagccagag caccggacag cacttccgtc tcttcatccg
     1261 ccacgggccg ttgcaggtga cggcagagga aggaggattc acctgttacg ggttgagcaa
     1321 aggaggtttc gttccctggt tcgagggcag aggaagtctg ctaacatgcg gtgacgtcga
     1381 ggagaatcct ggcccaatgg ataaaaagta ttctattggt ttagacatcg gcactaattc
     1441 cgttggatgg gctgtcataa ccgatgaata caaagtacct tcaaagaaat ttaaggtgtt
     1501 ggggaacaca gaccgtcatt cgattaaaaa gaatcttatc ggtgccctcc tattcgatag
     1561 tggcgaaacg gcagaggcga ctcgcctgaa acgaaccgct cggagaaggt atacacgtcg
     1621 caagaaccga atatgttact tacaagaaat ttttagcaat gagatggcca aagttgacga
     1681 ttctttcttt caccgtttgg aagagtcctt ccttgtcgaa gaggacaaga aacatgaacg
     1741 gcaccccatc tttggaaaca tagtagatga ggtggcatat catgaaaagt acccaacgat
     1801 ttatcacctc agaaaaaagc tagttgactc aactgataaa gcggacctga ggttaatcta
     1861 cttggctctt gcccatatga taaagttccg tgggcacttt ctcattgagg gtgatctaaa
     1921 tccggacaac tcggatgtcg acaaactgtt catccagtta gtacaaacct ataatcagtt
     1981 gtttgaagag aaccctataa atgcaagtgg cgtggatgcg aaggctattc ttagcgcccg
     2041 cctctctaaa tcccgacggc tagaaaacct gatcgcacaa ttacccggag agaagaaaaa
     2101 tgggttgttc ggtaacctta tagcgctctc actaggcctg acaccaaatt ttaagtcgaa
     2161 cttcgactta gctgaagatg ccaaattgca gcttagtaag gacacgtacg atgacgatct
     2221 cgacaatcta ctggcacaaa ttggagatca gtatgcggac ttatttttgg ctgccaaaaa
     2281 ccttagcgat gcaatcctcc tatctgacat actgagagtt aatactgaga ttaccaaggc
     2341 gccgttatcc gcttcaatga tcaaaaggta cgatgaacat caccaagact tgacacttct
     2401 caaggcccta gtccgtcagc aactgcctga gaaatataag gaaatattct ttgatcagtc
     2461 gaaaaacggg tacgcaggtt atattgacgg cggagcgagt caagaggaat tctacaagtt
     2521 tatcaaaccc atattagaga agatggatgg gacggaagag ttgcttgtaa aactcaatcg
     2581 cgaagatcta ctgcgaaagc agcggacttt cgacaacggt agcattccac atcaaatcca
     2641 cttaggcgaa ttgcatgcta tacttagaag gcaggaggat ttttatccgt tcctcaaaga
     2701 caatcgtgaa aagattgaga aaatcctaac ctttcgcata ccttactatg tgggacccct
     2761 ggcccgaggg aactctcggt tcgcatggat gacaagaaag tccgaagaaa cgattactcc
     2821 atggaatttt gaggaagttg tcgataaagg tgcgtcagct caatcgttca tcgagaggat
     2881 gaccaacttt gacaagaatt taccgaacga aaaagtattg cctaagcaca gtttacttta
     2941 cgagtatttc acagtgtaca atgaactcac gaaagttaag tatgtcactg agggcatgcg
     3001 taaacccgcc tttctaagcg gagaacagaa gaaagcaata gtagatctgt tattcaagac
     3061 caaccgcaaa gtgacagtta agcaattgaa agaggactac tttaagaaaa ttgaatgctt
     3121 cgattctgtc gagatctccg gggtagaaga tcgatttaat gcgtcacttg gtacgtatca
     3181 tgacctccta aagataatta aagataagga cttcctggat aacgaagaga atgaagatat
     3241 cttagaagat atagtgttga ctcttaccct ctttgaagat cgggaaatga ttgaggaaag
     3301 actaaaaaca tacgctcacc tgttcgacga taaggttatg aaacagttaa agaggcgtcg
     3361 ctatacgggc tggggacgat tgtcgcggaa acttatcaac gggataagag acaagcaaag
     3421 tggtaaaact attctcgatt ttctaaagag cgacggcttc gccaatagga actttatgca
     3481 gctgatccat gatgactctt taaccttcaa agaggatata caaaaggcac aggtttccgg
     3541 acaaggggac tcattgcacg aacatattgc gaatcttgct ggttcgccag ccatcaaaaa
     3601 gggcatactc cagacagtca aagtagtgga tgagctagtt aaggtcatgg gacgtcacaa
     3661 accggaaaac attgtaatcg agatggcacg cgaaaatcaa acgactcaga aggggcaaaa
     3721 aaacagtcga gagcggatga agagaataga agagggtatt aaagaactgg gcagccagat
     3781 cttaaaggag catcctgtgg aaaataccca attgcagaac gagaaacttt acctctatta
     3841 cctacaaaat ggaagggaca tgtatgttga tcaggaactg gacataaacc gtttatctga
     3901 ttacgacgtc gatcacattg taccccaatc ctttttgaag gacgattcaa tcgacaataa
     3961 agtgcttaca cgctcggata agaaccgagg gaaaagtgac aatgttccaa gcgaggaagt
     4021 cgtaaagaaa atgaagaact attggcggca gctcctaaat gcgaaactga taacgcaaag
     4081 aaagttcgat aacttaacta aagctgagag gggtggcttg tctgaacttg acaaggccgg
     4141 atttattaaa cgtcagctcg tggaaacccg ccaaatcaca aagcatgttg cacagatact
     4201 agattcccga atgaatacga aatacgacga gaacgataag ctgattcggg aagtcaaagt
     4261 aatcacttta aagtcaaaat tggtgtcgga cttcagaaag gattttcaat tctataaagt
     4321 tagggagata aataactacc accatgcgca cgacgcttat cttaatgccg tcgtagggac
     4381 cgcactcatt aagaaatacc cgaagctaga aagtgagttt gtgtatggtg attacaaagt
     4441 ttatgacgtc cgtaagatga tcgcgaaaag cgaacaggag ataggcaagg ctacagccaa
     4501 atacttcttt tattctaaca ttatgaattt ctttaagacg gaaatcactc tggcaaacgg
     4561 agagatacgc aaacgacctt taattgaaac caatggggag acaggtgaaa tcgtatggga
     4621 taagggccgg gacttcgcga cggtgagaaa agttttgtcc atgccccaag tcaacatagt
     4681 aaagaaaact gaggtgcaga ccggagggtt ttcaaaggaa tcgattcttc caaaaaggaa
     4741 tagtgataag ctcatcgctc gtaaaaagga ctgggacccg aaaaagtacg gtggcttcga
     4801 tagccctaca gttgcctatt ctgtcctagt agtggcaaaa gttgagaagg gaaaatccaa
     4861 gaaactgaag tcagtcaaag aattattggg gataacgatt atggagcgct cgtcttttga
     4921 aaagaacccc atcgacttcc ttgaggcgaa aggttacaag gaagtaaaaa aggatctcat
     4981 aattaaacta ccaaagtata gtctgtttga gttagaaaat ggccgaaaac ggatgttggc
     5041 tagcgccgga gagcttcaaa aggggaacga actcgcacta ccgtctaaat acgtgaattt
     5101 cctgtattta gcgtcccatt acgagaagtt gaaaggttca cctgaagata acgaacagaa
     5161 gcaacttttt gttgagcagc acaaacatta tctcgacgaa atcatagagc aaatttcgga
     5221 attcagtaag agagtcatcc tagctgatgc caatctggac aaagtattaa gcgcatacaa
     5281 caagcacagg gataaaccca tacgtgagca ggcggaaaat attatccatt tgtttactct
     5341 taccaacctc ggcgctccag ccgcattcaa gtattttgac acaacgatag atcgcaaacg
     5401 atacacttct accaaggagg tgctagacgc gacactgatt caccaatcca tcacgggatt
     5461 atatgaaact cggatagatt tgtcacagct tgggggtgac ggatccccca agaagaagag
     5521 gaaagtctcg agtagccgcg ctaagcgggc accggtgaaa cagactttga attttgacct
     5581 tctcaagttg gcgggagacg tggagtccaa cccagggccg gatatcatgg tgagcaaggg
     5641 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     5701 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     5761 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     5821 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     5881 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     5941 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     6001 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     6061 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     6121 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     6181 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     6241 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     6301 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaacagc tttcttgtac
     6361 aaagttggca ttataagaaa gcattgctta tcaatttgtt gcaacgaaca ggtcactatc
     6421 agtcaaaata aaatcattat ttgccatcca gctgcagctc tggcccgtgt ctcaaaatct
     6481 ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta
     6541 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg
     6601 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     6661 gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct
     6721 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     6781 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     6841 atggttactc accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc
     6901 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     6961 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     7021 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     7081 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     7141 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     7201 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     7261 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     7321 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
     7381 attggttaat tggttgtaac actggcagag cattacgctg acttgacggg acggcgcaag
     7441 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     7501 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     7561 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     7621 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     7681 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     7741 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     7801 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcg
//
