LOCUS       sequence-305026-        7354 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 7354)
  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 7354)
  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-eSpCas9n(1.1)-2A-EGFP-L2
FEATURES             Location/Qualifiers
     source          1..7354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             3854..3901
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3926..3991
                     /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(3998..4000,4001..4003,4004..4717)
                     /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(3998..4000,4001..4003,4004..4714)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4304..4323)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4651..4672
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4715..4814)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4926..4945)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4937..5746
                     /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(5004..5023)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5889..6477
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6378..6397
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6612..6639)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(6731..6817)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    6871..6966
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6990..7055
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             7062..7082
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(7107..7354,1..2290,2291..2293,2294..2755,2756..2758,
                     2759..2926,2927..2929,2930..3853)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
       61 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
      121 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
      181 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
      241 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
      301 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
      361 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
      421 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
      481 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
      541 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
      601 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
      661 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
      721 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
      781 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
      841 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
      901 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
      961 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     1021 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     1081 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     1141 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     1201 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     1261 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     1321 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     1381 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     1441 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     1501 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     1561 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     1621 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     1681 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     1741 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     1801 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     1861 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     1921 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     1981 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     2041 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     2101 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     2161 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     2221 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
     2281 gagctttctg gccgacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     2341 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     2401 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     2461 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     2521 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     2581 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     2641 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     2701 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctgcgct
     2761 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     2821 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     2881 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaaggcgc ctctgatcga
     2941 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     3001 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     3061 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     3121 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     3181 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     3241 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     3301 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     3361 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     3421 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     3481 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     3541 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     3601 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     3661 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     3721 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     3781 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     3841 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
     3901 ggaattcggc agtagccggg caccggtgaa acagactttg aattttgacc ttctcaagtt
     3961 ggcgggagac gtggagtcca acccagggcc ggatatcatg gtgagcaagg gcgaggagct
     4021 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     4081 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     4141 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     4201 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     4261 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     4321 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     4381 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     4441 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     4501 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     4561 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     4621 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     4681 cgggatcact ctcggcatgg acgagctgta caagtaacag ctttcttgta caaagttggc
     4741 attataagaa agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat
     4801 aaaatcatta tttgccatcc agctgcagct ctggcccgtg tctcaaaatc tctgatgtta
     4861 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag
     4921 taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc
     4981 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     5041 tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     5101 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     5161 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     5221 caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     5281 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     5341 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     5401 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     5461 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     5521 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     5581 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     5641 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     5701 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa
     5761 ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc
     5821 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5881 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5941 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6001 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6061 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6121 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6181 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6241 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6301 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6361 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6421 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6481 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6541 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgctagcca ggaagagttt
     6601 gtagaaacgc aaaaaggcca tccgtcagga tggccttctg cttagtttga tgcctggcag
     6661 tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg ttcaaatccg
     6721 ctcccggcgg atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa
     6781 aggcccagtc ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc
     6841 gcgttaacgc tagcatggat ctcgggcccc aaataatgat tttattttga ctgatagtga
     6901 cctgttcgtt gcaacaaatt gatgagcaat gcttttttat aatgccaact ttgtataata
     6961 aagttggctt cgaaggagat agaaccatgg actataagga ccacgacgga gactacaagg
     7021 atcatgatat tgattacaaa gacgatgacg ataagatggc cccaaagaag aagcggaagg
     7081 tcggtatcca cggagtccca gcagccgaca agaagtacag catcggcctg gccatcggca
     7141 ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca
     7201 aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt
     7261 tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca
     7321 ccagacggaa gaaccggatc tgctatctgc aaga
//
