LOCUS       sequence-79592-e        5338 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For in vitro transcription of Tol2 transposase using T7 Promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5338)
  AUTHORS   Khattak S, Murawala P, Andreas H, Kappert V, Schuez M, 
            Sandoval-Guzman T, Crawford K, Tanaka EM
  TITLE     Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, 
            metamorphosis, transgenesis and tamoxifen-mediated recombination.
  JOURNAL   Nat Protoc. 2014 Mar;9(3):529-40. doi: 10.1038/nprot.2014.040. Epub 
            2014 Feb 6.
  PUBMED    24504478
REFERENCE   2  (bases 1 to 5338)
  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"; doi: 
            "10.1038/nprot.2014.040"; journalName: "Nat Protoc"; date: 
            "2014-03"; volume: "9"; issue: "3"; pages: "529-40"
COMMENT     Sequence Label: T7-TPase
FEATURES             Location/Qualifiers
     source          1..5338
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     649..668
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        649..667
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             779..2728
                     /codon_start=1
                     /product="transposase of the Tol2 transposon from the 
                     medaka fish Oryzias latipes (Kawakami, 2007)"
                     /label=Tol2 transposase
                     /translation="MEEVCDSSAAASSTVQNQPQDQEHPWPYLREFFSLSGVNKDSFKM
                     KCVLCLPLNKEISAFKSSPSNLRKHIERMHPNYLKNYSKLTAQKRKIGTSTHASSSKQL
                     KVDSVFPVKHVSPVTVNKAILRYIIQGLHPFSTVDLPSFKELISTLQPGISVITRPTLR
                     SKIAEAALIMKQKVTAAMSEVEWIATTTDCWTARRKSFIGVTAHWINPGSLERHSAALA
                     CKRLMGSHTFEVLASAMNDIHSEYEIRDKVVCTTTDSGSNFMKAFRVFGVENNDIETEA
                     RRCESDDTDSEGCGEGSDGVEFQDASRVLDQDDGFEFQLPKHQKCACHLLNLVSSVDAQ
                     KALSNEHYKKLYRSVFGKCQALWNKSSRSALAAEAVESESRLQLLRPNQTRWNSTFMAV
                     DRILQICKEAGEGALRNICTSLEVPMFNPAEMLFLTEWANTMRPVAKVLDILQAETNTQ
                     LGWLLPSVHQLSLKLQRLHHSLRYCDPLVDALQQGIQTRFKHMFEDPEIIAAAILLPKF
                     RTSWTNDETIIKRGMDYIRVHLEPLDHKKELANSSSDDEDFFASLKPTTHEASKELDGY
                     LACVSDTRESLLTFPAICSLSIKTNTPLPASAACERLFSTAGLLFSPKRARLDTNNFEN
                     QLLLKLNLRFYNFE"
     primer_bind     complement(3013..3031)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3027..3074
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3127..3147)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3127..3145)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3189..3205)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3189..3205)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3202..3224)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3213..3229
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3237..3243,3244..3261,3262..3267))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3282..3303
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3420..3437)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3591..4179)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3671..3690)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4350..5141,5142..5210))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4973..4992
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5211..5315)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgtagggat aacagggtaa tgcgcgcgta atacgactca
      661 ctatagggcg aattgggtac cgggcccccc ctcgaggtcg agtcacatct attaccacaa
      721 tgcacagcac cttgacctgg aaattaggga aattataaca gtcaatcagt ggaagaaaat
      781 ggaggaagta tgtgattcat cagcagctgc gagcagcaca gtccaaaatc agccacagga
      841 tcaagagcac ccgtggccgt atcttcgcga attcttttct ttaagtggtg taaataaaga
      901 ttcattcaag atgaaatgtg tcctctgtct cccgcttaat aaagaaatat cggccttcaa
      961 aagttcgcca tcaaacctaa ggaagcatat tgagagaatg cacccaaatt acctcaaaaa
     1021 ctactctaaa ttgacagcac agaagagaaa gatcgggacc tccacccatg cttccagcag
     1081 taagcaactg aaagttgact cagttttccc agtcaaacat gtgtctccag tcactgtgaa
     1141 caaagctata ttaaggtaca tcattcaagg acttcatcct ttcagcactg ttgatctgcc
     1201 atcatttaaa gagctgatta gtacactgca gcctggcatt tctgtcatta caaggcctac
     1261 tttacgctcc aagatagctg aagctgctct gatcatgaaa cagaaagtga ctgctgccat
     1321 gagtgaagtt gaatggattg caaccacaac ggattgttgg actgcacgta gaaagtcatt
     1381 cattggtgta actgctcact ggatcaaccc tggaagtctt gaaagacatt ccgctgcact
     1441 tgcctgcaaa agattaatgg gctctcatac ttttgaggta ctggccagtg ccatgaatga
     1501 tatccactca gagtatgaaa tacgtgacaa ggttgtttgc acaaccacag acagtggttc
     1561 caactttatg aaggctttca gagtttttgg tgtggaaaac aatgatatcg agactgaggc
     1621 aagaaggtgt gaaagtgatg acactgattc tgaaggctgt ggtgagggaa gtgatggtgt
     1681 ggaattccaa gatgcctcac gagtcctgga ccaagacgat ggcttcgaat tccagctacc
     1741 aaaacatcaa aagtgtgcct gtcacttact taacctagtc tcaagcgttg atgcccaaaa
     1801 agctctctca aatgaacact acaagaaact ctacagatct gtctttggca aatgccaagc
     1861 tttatggaat aaaagcagcc gatcggctct agcagctgaa gctgttgaat cagaaagccg
     1921 gcttcagctt ttaaggccaa accaaacgcg gtggaattca acttttatgg ctgttgacag
     1981 aattcttcaa atttgcaaag aagcaggaga aggcgcactt cggaatatat gcacctctct
     2041 tgaggttcca atgtttaatc cagcagaaat gctgttcttg acagagtggg ccaacacaat
     2101 gcgtccagtt gcaaaagtac tcgacatctt gcaagcggaa acgaatacac agctggggtg
     2161 gctgctgcct agtgtccatc agttaagctt gaaacttcag cgactccacc attctctcag
     2221 gtactgtgac ccacttgtgg atgccctaca acaaggaatc caaacacgat tcaagcatat
     2281 gtttgaagat cctgagatca tagcagctgc catccttctc cctaaatttc ggacctcttg
     2341 gacaaatgat gaaaccatca taaaacgagg catggactac atcagagtgc atctggagcc
     2401 tttggaccac aagaaggaat tggccaacag ttcatctgat gatgaagatt ttttcgcttc
     2461 tttgaaaccg acaacacatg aagccagcaa agagttggat ggatatctgg cctgtgtttc
     2521 agacaccagg gagtctctgc tcacgtttcc tgctatttgc agcctctcta tcaagactaa
     2581 tacacctctt cccgcatcgg ctgcctgtga gaggcttttc agcactgcag gattgctttt
     2641 cagccccaaa agagctaggc ttgacactaa caattttgag aatcagcttc tactgaagtt
     2701 aaatctgagg ttttacaact ttgagtagcg tgtactggca ttagattgtc tgtcttatag
     2761 tttgataatt aaatacaaac agttctaaag caggataaaa ccttgtatgc atttcattta
     2821 atgttttttg agattaaaag cttaaacaag aaaaaaaaaa aaaaaaaaaa aaaaaagtac
     2881 tagtcgagcc tctagagcgg ccgccaccgc ggcgagacac ccggatcgcg ccaaaaaaaa
     2941 aaaaaaaaaa aaaaaaaaaa ccggatccgg ctgctaacaa agcccgaaag gaagctgagt
     3001 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     3061 tgaggggttt tttgctgaaa ggaggaacta tatccggata tcccgcaaga gctccagctt
     3121 ttgttccctt tagtgagggt taattgcgcg cattaccctg ttatccctac gcgcttggcg
     3181 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     3241 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3301 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3361 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3421 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3481 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3541 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3601 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3661 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3721 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3781 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3841 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3901 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3961 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4021 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4081 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4141 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4201 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4261 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4321 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4381 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4441 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4501 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4561 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4621 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4681 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4741 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4801 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4861 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4921 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4981 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5041 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5101 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5161 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5221 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5281 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
