LOCUS       sequence-273832-        4048 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 4048)
  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 4048)
  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-L5-CAG-R3
FEATURES             Location/Qualifiers
     source          1..4048
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        3..382
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        384..661
                     /label=chicken beta-actin promoter
     intron          663..1680
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1604..1626
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1688..1707
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1741..1864
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2001..2020)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2012..2821
                     /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(2079..2098)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2964..3552
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3453..3472
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3687..3714)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3806..3892)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    3946..4041
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 tcgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
       61 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      121 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      181 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      241 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      301 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      361 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
      421 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
      481 gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 ggggagtcgc tgcgacgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
      721 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
      781 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
      841 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag
      961 cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc
     1021 gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt
     1081 gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg
     1141 cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg
     1201 cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg
     1261 gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc
     1321 ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg
     1381 gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc
     1441 accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg
     1501 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt
     1561 ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt
     1621 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag
     1681 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcaagctt
     1741 caactttgta taataaagtt gaacgagaaa cgtaaaatga tataaatatc aatatattaa
     1801 attagatttt gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac
     1861 tatgaatcaa ctacttagat ggtattagtg acctgtactg cagctctggc ccgtgtctca
     1921 aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc
     1981 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag
     2041 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     2101 tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt
     2161 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     2221 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     2281 tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga
     2341 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
     2401 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
     2461 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     2521 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
     2581 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     2641 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     2701 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     2761 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     2821 atcagaattg gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg
     2881 cgcaagctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2941 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3001 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3061 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     3121 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3181 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3241 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3301 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3361 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3421 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3481 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3541 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3601 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgct
     3661 agccaggaag agtttgtaga aacgcaaaaa ggccatccgt caggatggcc ttctgcttag
     3721 tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca
     3781 caacgttcaa atccgctccc ggcggatttg tcctactcag gagagcgttc accgacaaac
     3841 aacagataaa acgaaaggcc cagtcttccg actgagcctt tcgttttatt tgatgcctgg
     3901 cagttcccta ctctcgcgtt aacgctagca tggatctcgg gccccaaata atgattttat
     3961 tttgactgat agtgacctgt tcgttgcaac aaattgatga gcaatgcttt tttataatgc
     4021 caactttgta tacaaaagtt gaagcttg
//
