LOCUS       sequence-167638-        5057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tol2 transposon with a puromycin-thymidine kinase fusion driven by a
            PGK promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5057)
  AUTHORS   Clark KJ, Carlson DF, Foster LK, Kong BW, Foster DN, Fahrenkrug SC
  TITLE     Enzymatic engineering of the porcine genome with transposons and 
            recombinases.
  JOURNAL   BMC Biotechnol. 2007 Jul 17;7:42.
  PUBMED    17640337
REFERENCE   2  (bases 1 to 5057)
  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: "BMC 
            Biotechnol."; date: "2007-07-17"; volume: "7"; pages: "42"
COMMENT     Sequence Label: pKTol2P-PTK
FEATURES             Location/Qualifiers
     source          1..5057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(10..21,22..209)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(231..250)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(232..250)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     658..677
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      739..748
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             745..1344
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MGTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPDSVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLGGDRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVECPKDRATWCMTRKAGA"
     primer_bind     1244..1264
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1351..2346
                     /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="MPSVLRVYIDGSHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     primer_bind     complement(2366..2383)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2372..2596
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    2662..2743
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    2867..2948
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        3049..3067
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    join(3087..3249,3250..3261)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3291..3308)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3462..4050)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3542..3561)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4193..5002)
                     /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     4916..4935
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4994..5013
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 gctggatccc agaggtgtaa agtacttgag taattttact tgattactgt acttaagtat
       61 tatttttggg gatttttact ttacttgagt acaattaaaa atcaatactt ttacttttac
      121 ttaattacat ttttttagaa aaaaaagtac tttttactcc ttacaatttt atttacagtc
      181 aaaaagtact tattttttgg agatcacttc attctatttt cccttggtct ccctatagtg
      241 agtcgtatta gatcccggga ttactagttt aaacctagga tgcataccgg tcaagctagc
      301 gatctgagat ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt
      361 tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg
      421 ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg
      481 tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat
      541 agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg
      601 ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat
      661 tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc
      721 tttcgacctg cagccaacgc caccatgggg accgagtaca agcccacggt gcgcctcgcc
      781 acccgcgacg acgtcccccg ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
      841 gccacgcgcc acaccgtcga cccggaccgc cacatcgagc gggtcaccga gctgcaagaa
      901 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
      961 gcggtggcgg tctggaccac gccggacagc gtcgaagcgg gggcggtgtt cgccgagatc
     1021 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     1081 ctcctgggcg gcgaccgacc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     1141 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     1201 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     1261 gagcggctcg gcttcaccgt caccgccgac gtcgagtgcc cgaaggaccg cgcgacctgg
     1321 tgcatgaccc gcaaggccgg tgccggatcc atgcccagcg tactgcgggt ttatatagac
     1381 ggttctcacg ggatggggaa aaccaccacc acgcaactgc tggtggccct gggttcgcgc
     1441 gacgatatcg tctacgtacc cgagccgatg acttactggc aggtgctggg ggcttccgag
     1501 acaatcgcga acatctacac cacacaacac cgcctcgacc agggtgagat atcggccggg
     1561 gacgcggcgg tggtaatgac aagcgcccag ataacaatgg gcatgcctta tgccgtgacc
     1621 gacgccgttc tggctcctca tatcgggggg gaggctggga gctcacatgc cccgcccccg
     1681 gccctcaccc tcatcttcga ccgccatccc atcgccgccc tcctgtgcta cccggccgcg
     1741 cgatacctta tgggcagcat gaccccccag gccgtgctgg cgttcgtggc cctcatcccg
     1801 ccgaccttgc ccggcacaaa catcgtgttg ggggcccttc cggaggacag acacatcgac
     1861 cgcctggcca aacgccagcg ccccggcgag cggcttgacc tggctatgct ggccgcgatt
     1921 cgccgcgttt acgggctgct tgccaatacg gtgcggtatc tgcagggcgg cgggtcgtgg
     1981 cgggaggatt ggggacagct ttcggggacg gccgtgccgc cccagggtgc cgagccccag
     2041 agcaacgcgg gcccacgacc ccatatcggg gacacgttat ttaccctgtt tcgggccccc
     2101 gagttgctgg cccccaacgg cgacctgtac aacgtgtttg cctgggcctt ggacgtcttg
     2161 gccaaacgcc tccgtcccat gcacgtcttt atcctggatt acgaccaatc gcccgccggc
     2221 tgccgggacg ccctgctgca acttacctcc gggatggtcc agacccacgt caccaccccc
     2281 ggctccatac cgacgatctg cgacctggcg cgcacgtttg cccgggagat gggggaggct
     2341 aactgatcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     2401 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2461 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2521 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2581 gcggtgggct ctatggcttc tgaggcggaa agaaccaaag ggcgaattca ttgatgagtt
     2641 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     2701 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     2761 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     2821 ctacaaatgt ggtcattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     2881 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     2941 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     3001 aggtttttta aagcaagtaa aacctctaca aatgtggtca ttcagatcaa ttaaccctca
     3061 ctaaagggag accactctag ttttctttct tgcttttact tttacttcct taatactcaa
     3121 gtacaatttt aatggagtac ttttttactt ttactcaagt aagattctag ccagatactt
     3181 ttacttttaa ttgagtaaaa ttttccctaa gtacttgtac tttcacttga gtaaaatttt
     3241 tgagtacttt ttacacctct gctcggtacc atttaaatct gttccgcttc ctcgctcact
     3301 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3361 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3421 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3481 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3541 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3601 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa ccgtggcgct ttctcatagc
     3661 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3721 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3781 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3841 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3901 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3961 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4021 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4081 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4141 atcttcacct agatcctttt tgccagtgtt acaaccaatt aaccaattct gattagaaaa
     4201 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     4261 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     4321 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     4381 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     4441 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     4501 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     4561 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     4621 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     4681 atacctggaa tgctgttttt ccggggatcg cagtggtgag taaccatgca tcatcaggag
     4741 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     4801 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     4861 gcgcatcggg cttcccatac aagcgataga ttgtcgcacc tgattgcccg acattatcgc
     4921 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctcgacg
     4981 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     5041 ttattgttca tgatgca
//
