LOCUS       sequence-304877-        3627 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 3627)
  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 3627)
  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-EF1_-R3
FEATURES             Location/Qualifiers
     source          1..3627
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        86..1264
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          317..1255
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1212..1232
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1281..1300
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1281..1299
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1326..1449
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1586..1605)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1597..2406
                     /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(1664..1683)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2549..3137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3038..3057
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3272..3299)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3391..3477)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    3531..3626
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 agctttgcaa agatggataa agttttaaac agagaggaat ctttgcagct aatggacctt
       61 ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc
      121 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
      181 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
      241 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
      301 tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg
      361 ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc
      421 cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc
      481 gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg
      541 gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg
      601 acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca
      661 cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac
      721 atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca
      781 agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc
      841 ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc
      901 tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc
      961 cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta
     1021 ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg
     1081 ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt
     1141 taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc
     1201 ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc
     1261 gtgaggaatt agcttggtac taatacgact cactataggg agacccaagc tggctaggta
     1321 agcttcaact ttgtataata aagttgaacg agaaacgtaa aatgatataa atatcaatat
     1381 attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca
     1441 gtcactatga atcaactact tagatggtat tagtgacctg tactgcagct ctggcccgtg
     1501 tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg aacaataaaa
     1561 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg
     1621 tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
     1681 gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca
     1741 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
     1801 agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact
     1861 cctgatgatg catggttact caccactgcg atccccggaa aaacagcatt ccaggtatta
     1921 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg
     1981 ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct
     2041 caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
     2101 aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg
     2161 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     2221 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     2281 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     2341 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     2401 ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct gacttgacgg
     2461 gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2521 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2581 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2641 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     2701 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2761 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2821 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2881 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     2941 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3001 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3061 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3121 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3181 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3241 ccgctagcca ggaagagttt gtagaaacgc aaaaaggcca tccgtcagga tggccttctg
     3301 cttagtttga tgcctggcag tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg
     3361 cttcacaacg ttcaaatccg ctcccggcgg atttgtccta ctcaggagag cgttcaccga
     3421 caaacaacag ataaaacgaa aggcccagtc ttccgactga gcctttcgtt ttatttgatg
     3481 cctggcagtt ccctactctc gcgttaacgc tagcatggat ctcgggcccc aaataatgat
     3541 tttattttga ctgatagtga cctgttcgtt gcaacaaatt gatgagcaat gcttttttat
     3601 aatgccaact ttgtatacaa aagttga
//
