LOCUS       sequence-305032-        5225 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 5225)
  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 5225)
  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-L1-PGK-HygTK-L2
FEATURES             Location/Qualifiers
     source          1..5225
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(43..129)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    182..281
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        288..787
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(323..346)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     720..739
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1891..2886
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRSMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     protein_bind    complement(3252..3351)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3463..3482)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3474..4283
                     /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(3541..3560)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4426..5014
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4915..4934
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5149..5176)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag
       61 agcgttcacc gacaaacaac agataaaacg aaaggcccag tcttccgact gagcctttcg
      121 ttttatttga tgcctggcag ttccctactc tcgcgttaac gctagcatgg atctcgggcc
      181 ccaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca
      241 atgctttttt ataatgccaa ctttgtacaa aaaagcagaa ttctaccggg taggggaggc
      301 gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta
      361 cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc
      421 gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc
      481 cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct
      541 cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca
      601 atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg
      661 cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc
      721 attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg
      781 cctttcgacc gatcatcaag cttgatcctc atgaaaaagc ctgaactcac cgcgacgtct
      841 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
      901 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
      961 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     1021 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     1081 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     1141 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     1201 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     1261 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     1321 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     1381 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     1441 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     1501 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     1561 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     1621 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     1681 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     1741 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tcgcgtctgc gttcgaccag
     1801 gctgcgcgtt ctcgcggcca tagcaaccga cgtacggcgt tgcgccctcg ccggcagcaa
     1861 gaagccacgg aagtccgccc ggagcagaaa atgcccacgc tactgcgggt ttatatagac
     1921 ggtccccacg ggatggggaa aaccaccacc acgcaactgc tggtggccct gggttcgcgc
     1981 gacgatatcg tctacgtacc cgagccgatg acttactggc gggtgctggg ggcttccgag
     2041 acaatcgcga acatctacac cacacaacac cgcctcgacc agggtgagat atcggccggg
     2101 gacgcggcgg tggtaatgac aagcgcccag ataacaatgg gcatgcctta tgccgtgacc
     2161 gacgccgttc tggctcctca tatcgggggg gaggctggga gctcacatgc cccgcccccg
     2221 gccctcaccc tcatcttcga ccgccatccc atcgccgccc tcctgtgcta cccggccgcg
     2281 cggtacctta tgggcagcat gaccccccag gccgtgctgg cgttcgtggc cctcatcccg
     2341 ccgaccttgc ccggcaccaa catcgtgctt ggggcccttc cggaggacag acacatcgac
     2401 cgcctggcca aacgccagcg ccccggcgag cggctggacc tggctatgct ggctgcgatt
     2461 cgccgcgttt acgggctact tgccaatacg gtgcggtatc tgcagtgcgg cgggtcgtgg
     2521 cgggaggact ggggacagct ttcggggacg gccgtgccgc cccagggtgc cgagccccag
     2581 agcaacgcgg gcccacgacc ccatatcggg gacacgttat ttaccctgtt tcgggccccc
     2641 gagttgctgg cccccaacgg cgacctgtat aacgtgtttg cctgggcctt ggacgtcttg
     2701 gccaaacgcc tccgttccat gcacgtcttt atcctggatt acgaccaatc gcccgccggc
     2761 tgccgggacg ccctgctgca acttacctcc gggatggtcc agacccacgt caccaccccc
     2821 ggctccatac cgacgatatg cgacctggcg cgcacgtttg cccgggagat gggggaggct
     2881 aactgagctc tagcttatcg actcgccgat agtggaaacc gacgccccag cactcgtccg
     2941 agggcaaagg aatagtcgag aaattgatga tctattaaac aataaagatg tccacatgga
     3001 agtttttcct gtcatacttt gttaagaagg gtgagaacag agtacctaca ttttgaatgg
     3061 aaggattgga gctacggggg tgggggtggg gtgggattag ataaatgcct gctctttact
     3121 gaaggctctt tactattgct ttatgataat gtttcatagt tggatatcat aatttaaaca
     3181 agcaaaacca aattaagggc cagctcattc ctcccactca tgatctatag atcctctaga
     3241 gtcgagggct gcagcagctt tcttgtacaa agttggcatt ataagaaagc attgcttatc
     3301 aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt gccatccagc
     3361 tgcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat
     3421 catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
     3481 atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct gatttatatg
     3541 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg
     3601 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     3661 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     3721 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggaaaaa
     3781 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     3841 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     3901 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     3961 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac
     4021 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     4081 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     4141 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     4201 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     4261 tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac tggcagagca
     4321 ttacgctgac ttgacgggac ggcgcaagct catgaccaaa atcccttaac gtgagttttc
     4381 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4441 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4501 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4561 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4621 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4681 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4741 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4801 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4861 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4921 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4981 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5041 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     5101 tgtggataac cgtattaccg ctagccagga agagtttgta gaaacgcaaa aaggccatcc
     5161 gtcaggatgg ccttctgctt agtttgatgc ctggcagttt atggcgggcg tcctgcccgc
     5221 caccc
//
