LOCUS       sequence-302045-        7219 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 7219)
  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 7219)
  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-SpCas9-HF4-2A-EGFP-L2
FEATURES             Location/Qualifiers
     source          1..7219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(2..1348,1349..1351,1352..1489,1490..1492,1493..1981,
                     1982..1984,1985..2083,2084..2086,2087..2776,2777..2779,
                     2780..4105)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Kleinstiver et al., 2016)"
                     /label=SpCas9-HF4
                     /note="carries the mutations Y450A, N497A, R661A, Q695A, 
                     and Q926A"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPAYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4112..4132
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4160..4225
                     /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(4232..4234,4235..4237,4238..4951)
                     /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(4232..4234,4235..4237,4238..4948)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4277..4298)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4538..4557)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4885..4906
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4949..5048)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5160..5179)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5171..5980
                     /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(5238..5257)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6123..6711
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6612..6631
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6846..6873)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(6965..7051)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    7105..7200
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 catggataaa aagtattcta ttggtttaga catcggcact aattccgttg gatgggctgt
       61 cataaccgat gaatacaaag taccttcaaa gaaatttaag gtgttgggga acacagaccg
      121 tcattcgatt aaaaagaatc ttatcggtgc cctcctattc gatagtggcg aaacggcaga
      181 ggcgactcgc ctgaaacgaa ccgctcggag aaggtataca cgtcgcaaga accgaatatg
      241 ttacttacaa gaaattttta gcaatgagat ggccaaagtt gacgattctt tctttcaccg
      301 tttggaagag tccttccttg tcgaagagga caagaaacat gaacggcacc ccatctttgg
      361 aaacatagta gatgaggtgg catatcatga aaagtaccca acgatttatc acctcagaaa
      421 aaagctagtt gactcaactg ataaagcgga cctgaggtta atctacttgg ctcttgccca
      481 tatgataaag ttccgtgggc actttctcat tgagggtgat ctaaatccgg acaactcgga
      541 tgtcgacaaa ctgttcatcc agttagtaca aacctataat cagttgtttg aagagaaccc
      601 tataaatgca agtggcgtgg atgcgaaggc tattcttagc gcccgcctct ctaaatcccg
      661 acggctagaa aacctgatcg cacaattacc cggagagaag aaaaatgggt tgttcggtaa
      721 ccttatagcg ctctcactag gcctgacacc aaattttaag tcgaacttcg acttagctga
      781 agatgccaaa ttgcagctta gtaaggacac gtacgatgac gatctcgaca atctactggc
      841 acaaattgga gatcagtatg cggacttatt tttggctgcc aaaaacctta gcgatgcaat
      901 cctcctatct gacatactga gagttaatac tgagattacc aaggcgccgt tatccgcttc
      961 aatgatcaaa aggtacgatg aacatcacca agacttgaca cttctcaagg ccctagtccg
     1021 tcagcaactg cctgagaaat ataaggaaat attctttgat cagtcgaaaa acgggtacgc
     1081 aggttatatt gacggcggag cgagtcaaga ggaattctac aagtttatca aacccatatt
     1141 agagaagatg gatgggacgg aagagttgct tgtaaaactc aatcgcgaag atctactgcg
     1201 aaagcagcgg actttcgaca acggtagcat tccacatcaa atccacttag gcgaattgca
     1261 tgctatactt agaaggcagg aggattttta tccgttcctc aaagacaatc gtgaaaagat
     1321 tgagaaaatc ctaacctttc gcatacctgc ctatgtggga cccctggccc gagggaactc
     1381 tcggttcgca tggatgacaa gaaagtccga agaaacgatt actccctgga attttgagga
     1441 agttgtcgat aaaggtgcgt cagctcaatc gttcatcgag aggatgaccg cctttgacaa
     1501 gaatttaccg aacgaaaaag tattgcctaa gcacagttta ctttacgagt atttcacagt
     1561 gtacaatgaa ctcacgaaag ttaagtatgt cactgagggc atgcgtaaac ccgcctttct
     1621 aagcggagaa cagaagaaag caatagtaga tctgttattc aagaccaacc gcaaagtgac
     1681 agttaagcaa ttgaaagagg actactttaa gaaaattgaa tgcttcgatt ctgtcgagat
     1741 ctccggggta gaagatcgat ttaatgcgtc acttggtacg tatcatgacc tcctaaagat
     1801 aattaaagat aaggacttcc tggataacga agagaatgaa gatatcttag aagatatagt
     1861 gttgactctt accctctttg aagatcggga aatgattgag gaaagactaa aaacatacgc
     1921 tcacctgttc gacgataagg ttatgaaaca gttaaagagg cgtcgctata cgggctgggg
     1981 agccttgtcg cggaaactta tcaacgggat aagagacaag caaagtggta aaactattct
     2041 cgattttcta aagagcgacg gcttcgccaa taggaacttt atggccctga tccatgatga
     2101 ctctttaacc ttcaaagagg atatacaaaa ggcacaggtt tccggacaag gggactcatt
     2161 gcacgaacat attgcgaatc ttgctggttc gccagccatc aaaaagggca tactccagac
     2221 agtcaaagta gtggatgagc tagttaaggt catgggacgt cacaaaccgg aaaacattgt
     2281 aatcgagatg gcacgcgaaa atcaaacgac tcagaagggg caaaaaaaca gtcgagagcg
     2341 gatgaagaga atagaagagg gtattaaaga actgggcagc cagatcttaa aggagcatcc
     2401 tgtggaaaat acccaattgc agaacgagaa actttacctc tattacctac aaaatggaag
     2461 ggacatgtat gttgatcagg aactggacat aaaccgttta tctgattacg acgtcgatca
     2521 cattgtaccc caatcctttt tgaaggacga ttcaatcgac aataaagtgc ttacacgctc
     2581 ggataagaac cgagggaaaa gtgacaatgt tccaagcgag gaagtcgtaa agaaaatgaa
     2641 gaactattgg cggcagctcc taaatgcgaa actgataacg caaagaaagt tcgataactt
     2701 aactaaagct gagaggggtg gcttgtctga acttgacaag gccggattta ttaaacgtca
     2761 gctcgtggaa acccgcgcca tcacaaagca tgttgcgcag atactagatt cccgaatgaa
     2821 tacgaaatac gacgagaacg ataagctgat tcgggaagtc aaagtaatca ctttaaagtc
     2881 aaaattggtg tcggacttca gaaaggattt tcaattctat aaagttaggg agataaataa
     2941 ctaccaccat gcgcacgacg cttatcttaa tgccgtcgta gggaccgcac tcattaagaa
     3001 atacccgaag ctagaaagtg agtttgtgta tggtgattac aaagtttatg acgtccgtaa
     3061 gatgatcgcg aaaagcgaac aggagatagg caaggctaca gccaaatact tcttttattc
     3121 taacattatg aatttcttta agacggaaat cactctggca aacggagaga tacgcaaacg
     3181 acctttaatt gaaaccaatg gggagacagg tgaaatcgta tgggataagg gccgggactt
     3241 cgcgacggtg agaaaagttt tgtccatgcc ccaagtcaac atagtaaaga aaactgaggt
     3301 gcagaccgga gggttttcaa aggaatcgat tcttccaaaa aggaatagtg ataagctcat
     3361 cgctcgtaaa aaggactggg acccgaaaaa gtacggtggc ttcgatagcc ctacagttgc
     3421 ctattctgtc ctagtagtgg caaaagttga gaagggaaaa tccaagaaac tgaagtcagt
     3481 caaagaatta ttggggataa cgattatgga gcgctcgtct tttgaaaaga accccatcga
     3541 cttccttgag gcgaaaggtt acaaggaagt aaaaaaggat ctcataatta aactaccaaa
     3601 gtatagtctg tttgagttag aaaatggccg aaaacggatg ttggctagcg ccggagagct
     3661 tcaaaagggg aacgaactcg cactaccgtc taaatacgtg aatttcctgt atttagcgtc
     3721 ccattacgag aagttgaaag gttcacctga agataacgaa cagaagcaac tttttgttga
     3781 gcagcacaaa cattatctcg acgaaatcat agagcaaatt tcggaattca gtaagagagt
     3841 catcctagct gatgccaatc tggacaaagt attaagcgca tacaacaagc acagggataa
     3901 acccatacgt gagcaggcgg aaaatattat ccatttgttt actcttacca acctcggcgc
     3961 tccagccgca ttcaagtatt ttgacacaac gatagatcgc aaacgataca cttctaccaa
     4021 ggaggtgcta gacgcgacac tgattcacca atccatcacg ggattatatg aaactcggat
     4081 agatttgtca cagcttgggg gtgacggatc ccccaagaag aagaggaaag tctcgagtag
     4141 ccgcgctaag cgggcaccgg tgaaacagac tttgaatttt gaccttctca agttggcggg
     4201 agacgtggag tccaacccag ggccggatat catggtgagc aagggcgagg agctgttcac
     4261 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     4321 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     4381 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     4441 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     4501 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     4561 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     4621 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     4681 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     4741 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     4801 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     4861 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     4921 cactctcggc atggacgagc tgtacaagta acagctttct tgtacaaagt tggcattata
     4981 agaaagcatt gcttatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc
     5041 attatttgcc atccagctgc agctctggcc cgtgtctcaa aatctctgat gttacattgc
     5101 acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac
     5161 aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat
     5221 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     5281 aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     5341 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     5401 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     5461 tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     5521 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     5581 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     5641 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     5701 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     5761 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     5821 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     5881 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     5941 taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt
     6001 gtaacactgg cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc
     6061 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6121 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6181 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6241 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6301 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6361 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6421 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6481 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6541 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6601 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6661 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6721 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6781 gcgttatccc ctgattctgt ggataaccgt attaccgcta gccaggaaga gtttgtagaa
     6841 acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg
     6901 gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg
     6961 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
     7021 agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcgtta
     7081 acgctagcat ggatctcggg ccccaaataa tgattttatt ttgactgata gtgacctgtt
     7141 cgttgcaaca aattgatgag caatgctttt ttataatgcc aactttgtat aataaagttg
     7201 gcttcgaagg agatagaac
//
