LOCUS       sequence-301972-        4046 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 4046)
  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 4046)
  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..4046
                     /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          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1739..1862
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1999..2018)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2010..2819
                     /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(2077..2096)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2962..3550
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3451..3470
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3685..3712)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3804..3890)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    3944..4039
                     /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 gccrggsggg gsggggsggg gsgrggggsg
      541 gggsggggsg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtac cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg rgggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcaagcttca
     1741 actttgtata ataaagttga acgagaaacg taaaatgata taaatatcaa tatattaaat
     1801 tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat ccagtcacta
     1861 tgaatcaact acttagatgg tattagtgac ctgtactgca gctctggccc gtgtctcaaa
     1921 atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg
     1981 cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc
     2041 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     2101 tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt
     2161 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
     2221 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     2281 atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat
     2341 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     2401 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
     2461 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     2521 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
     2581 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     2641 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     2701 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     2761 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat
     2821 cagaattggt taattggttg taacactggc agagcattac gctgacttga cgggacggcg
     2881 caagctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2941 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3001 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3061 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     3121 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3181 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3241 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3301 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3361 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3421 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3481 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3541 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3601 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgctag
     3661 ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt
     3721 tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctcctggccg ttgcttcaca
     3781 acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa
     3841 cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg atgcctggca
     3901 gttccctact ctcgcgttaa cgctagcatg gatctcgggc cccaaataat gattttattt
     3961 tgactgatag tgacctgttc gttgcaacaa attgatgagc aatgcttttt tataatgcca
     4021 actttgtata caaaagttga agcttg
//
