LOCUS       sequence-165953-        4450 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tol2 transposon with EGFP driven by minimal-CAGs promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4450)
  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 4450)
  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: pKTol2C-EGFP
FEATURES             Location/Qualifiers
     source          1..4450
                     /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"
     enhancer        336..639
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        641..916
                     /label=chicken beta-actin promoter
     primer_bind     1172..1191
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(1238..1240,1241..1243,1244..1957)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1238..1240,1241..1243,1244..1954)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1283..1304)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1544..1563)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1891..1912
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1961..1980)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2026..2081
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2080..2099)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        2442..2460
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    join(2480..2642,2643..2654)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(2684..2701)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2855..3443)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2935..2954)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3586..4395)
                     /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     4309..4328
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4387..4406
                     /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 aaacctagtt attaatagta atcaattacg
      301 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      361 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      421 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      481 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      541 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      601 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
      661 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
      721 taattatttt gtgcagcgat gggggcgggg gggggggggg cgcgcgccag gcggggcggg
      781 gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg
      841 cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg
      901 aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc
      961 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
     1021 ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt
     1081 tctgtggctg cgtgaaagcc ttgaggggct ccgggagcta gagcctctgc taaccatgtt
     1141 catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat
     1201 cattttggca aagaattcct cgagcctgaa ttctaccatg gtgagcaagg gcgaggagct
     1261 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1321 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     1381 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     1441 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1501 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1561 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1621 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     1681 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     1741 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1801 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     1861 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     1921 cgggatcact ctcggcatgg acgagctgta caagtaaaga tctttttccc tctgccaaaa
     1981 attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta
     2041 ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga
     2101 gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc
     2161 atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc
     2221 cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta
     2281 tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac
     2341 tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat
     2401 ggagatccct cgacctgcat accggtcaag ctagcgatat caattaaccc tcactaaagg
     2461 gagaccactc tagttttctt tcttgctttt acttttactt ccttaatact caagtacaat
     2521 tttaatggag tactttttta cttttactca agtaagattc tagccagata cttttacttt
     2581 taattgagta aaattttccc taagtacttg tactttcact tgagtaaaat ttttgagtac
     2641 tttttacacc tctgctcggt accatttaaa tctgttccgc ttcctcgctc actgactcgc
     2701 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2761 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2821 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2881 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2941 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3001 ccggatacct gtccgccttt ctcccttcgg gaaccgtggc gctttctcat agctcacgct
     3061 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3121 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3181 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3241 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     3301 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3361 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3421 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3481 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3541 cctagatcct ttttgccagt gttacaacca attaaccaat tctgattaga aaaactcatc
     3601 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     3661 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     3721 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     3781 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     3841 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     3901 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     3961 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     4021 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     4081 gaatgctgtt tttccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     4141 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     4201 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     4261 gggcttccca tacaagcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     4321 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg acgtttcccg
     4381 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     4441 tcatgatgca
//
