LOCUS       sequence-302034-        6550 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 6550)
  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 6550)
  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-Cas9n-L2
FEATURES             Location/Qualifiers
     source          1..6550
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(199..218)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             210..1019
                     /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(277..296)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1162..1750
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1651..1670
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1885..1912)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2004..2090)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    2144..2239
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2263..2328
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2335..2355
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2380..2403,2404..2406,2407..6480)
                     /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             2380..6480
                     /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             6481..6528
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    complement(join(6538..6550,1..87))
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 gtacaaagtt ggcattataa gaaagcattg cttatcaatt tgttgcaacg aacaggtcac
       61 tatcagtcaa aataaaatca ttatttgcca tccagctgca gctctggccc gtgtctcaaa
      121 atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg
      181 cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc
      241 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
      301 tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt
      361 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
      421 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
      481 atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat
      541 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
      601 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
      661 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
      721 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
      781 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
      841 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
      901 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
      961 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat
     1021 cagaattggt taattggttg taacactggc agagcattac gctgacttga cgggacggcg
     1081 caagctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     1141 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     1201 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     1261 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     1321 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     1381 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     1441 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     1501 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     1561 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     1621 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     1681 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     1741 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     1801 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgctag
     1861 ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt
     1921 tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca
     1981 acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa
     2041 cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg atgcctggca
     2101 gttccctact ctcgcgttaa cgctagcatg gatctcgggc cccaaataat gattttattt
     2161 tgactgatag tgacctgttc gttgcaacaa attgatgagc aatgcttttt tataatgcca
     2221 actttgtata ataaagttgg cttcgaagga gatagaacca tggactataa ggaccacgac
     2281 ggagactaca aggatcatga tattgattac aaagacgatg acgataagat ggccccaaag
     2341 aagaagcgga aggtcggtat ccacggagtc ccagcagccg acaagaagta cagcatcggc
     2401 ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc
     2461 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc
     2521 ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc
     2581 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac
     2641 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa
     2701 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac
     2761 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag
     2821 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc
     2881 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg
     2941 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc
     3001 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag
     3061 ctgcccggcg agaagaagaa tggcctgttc ggcaacctga ttgccctgag cctgggcctg
     3121 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag
     3181 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac
     3241 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
     3301 aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac
     3361 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa
     3421 gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc
     3481 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa
     3541 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc
     3601 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat
     3661 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc
     3721 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag
     3781 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc
     3841 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg
     3901 cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa
     3961 tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc
     4021 gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac
     4081 ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac
     4141 gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac
     4201 aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac
     4261 agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg
     4321 aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac
     4381 ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc
     4441 gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc
     4501 cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc
     4561 ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg
     4621 aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag
     4681 accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc
     4741 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac
     4801 gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg
     4861 gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag
     4921 gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac
     4981 aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac
     5041 gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg
     5101 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca
     5161 aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag
     5221 ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag
     5281 gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac
     5341 ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc
     5401 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa
     5461 atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc
     5521 gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa
     5581 accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc
     5641 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag
     5701 tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct
     5761 aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa
     5821 gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc
     5881 atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa
     5941 gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac
     6001 ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg
     6061 ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc
     6121 cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag
     6181 atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac
     6241 aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat
     6301 atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac
     6361 accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc
     6421 caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac
     6481 aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagga attctaatga
     6541 cagctttctt
//
