LOCUS       sequence-312935-        6018 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Tol2 transposase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6018)
  AUTHORS   Heinz MC, Oost KC, Snippert HJG
  TITLE     Introducing the Stem Cell ASCL2 Reporter STAR into Intestinal 
            Organoids.
  JOURNAL   STAR Protoc. 2020 Oct 8;1(3):100126. doi: 
            10.1016/j.xpro.2020.100126. eCollection 2020 Dec 18.
  PUBMED    33377020
REFERENCE   2  (bases 1 to 6018)
  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: "STAR 
            Protoc."; date: "2020-10-8"; volume: "1(3):100126. doi"; pages: " 
            10.1016/j.xpro.2020.100126. eCollection 2020 Dec 18"
COMMENT     Sequence Label: CMV-Tol2 transposase
FEATURES             Location/Qualifiers
     source          1..6018
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..978
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1013..1031
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1013..1030
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      1063..1072
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1069..3018
                     /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"
     polyA_signal    3048..3182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3072..3091)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3126..3145
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3295..3315)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3295..3313)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3334..3350)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3334..3350)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3347..3369)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3358..3374
                     /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(3382..3388,3389..3406,3407..3412))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3427..3448
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3565..3582)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3736..4324)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3816..3835)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4495..5286,5287..5355))
                     /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     5118..5137
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5356..5460)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5486..5941)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5623..5644)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5835..5854
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
       61 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
      121 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt
      181 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
      241 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
      301 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
      361 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
      421 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
      481 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
      541 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
      601 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
      661 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
      721 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
      781 tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
      841 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
      901 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
      961 aatgctcgtt tagggaaccg ccattctgcc tggggacgtc ggagcaagct tgatttaggt
     1021 gacactatag aatacaagct acttgttctt tttgcaggat ccgccaccat ggaagaggtg
     1081 tgcgactctt ccgccgcagc ttcatctacc gtccagaacc agcctcagga ccaagagcac
     1141 ccctggccat atctgagaga gttcttctcc ctgagcggag tgaataagga ctcatttaaa
     1201 atgaagtgtg tgctgtgtct tccactcaac aaagagatta gcgcgtttaa atcctcaccc
     1261 tcgaacctga ggaagcacat tgagcgtatg caccctaact acctgaaaaa ttactccaag
     1321 ctgaccgccc agaagagaaa gataggcact agtactcacg catccagcag taagcagttg
     1381 aaagtagact cagtgttccc tgttaagcat gtgtcccctg taacagttaa caaggctatt
     1441 ctccggtaca ttatccaagg tctgcacccg ttcagcaccg tcgacctacc ctccttcaag
     1501 gagctgatta gtacgcttca gcctggaatt agtgtgatta ctcgccctac tctgagaagc
     1561 aaaatcgcag aagcggcgct gatcatgaaa cagaaagtga ctgctgcaat gagcgaggtg
     1621 gaatggatcg cgacgacaac cgactgttgg acagcacgta ggaagtcctt tataggcgtc
     1681 actgcccact ggattaaccc cggttcactg gagcgacact cagcagccct ggcctgcaaa
     1741 cgccttatgg gatctcacac ttttgaggtg ttagcgtctg ctatgaacga catccactcg
     1801 gaatacgaaa ttagggacaa ggtggtctgt accacaacag actctggatc taactttatg
     1861 aaagcgtttc gcgtttttgg cgtggagaac aatgatatcg aaacggaggc cagacgctgt
     1921 gagtcagacg acactgacag cgaaggctgc ggcgaaggtt cagacggcgt cgaatttcag
     1981 gacgcgagtc gcgttctcga ccaggatgat ggatttgaat ttcagttacc aaagcaccag
     2041 aagtgtgcct gtcatcttct aaacctcgtc tcttcggtcg acgcacaaaa agctctgtcc
     2101 aacgagcatt acaagaagct ctaccggtct gtcttcggca agtgtcaggc cctttggaac
     2161 aagtcctcca gatccgctct cgccgcagag gcggtagaga gtgagtccag acttcagctg
     2221 ctgagaccta atcagacacg ctggaacagc actttcatgg cagtggaccg aatcttacaa
     2281 atctgtaaag aagccggtga gggcgctctc cgcaacatct gtacatcact ggaagtccct
     2341 atgtttaacc cagctgagat gctcttcttg acagagtggg ctaacactat gcggccagtc
     2401 gctaaagtcc tcgacatcct tcaggccgaa accaatacac agttaggctg gctccttcca
     2461 agcgtgcacc aactcagtct gaagctccaa agactgcatc atagtctgcg gtactgcgat
     2521 cccctcgtgg atgctctgca gcagggaatc cagactcggt tcaaacacat gttcgaagac
     2581 cctgagatta tcgctgcagc catccttctc ccaaaatttc gaacctcgtg gacaaacgat
     2641 gagaccatta ttaagcgcgg catggattat atccgcgtcc accttgaacc cctggaccac
     2701 aaaaaagagc tggctaattc aagtagtgac gatgaggatt tctttgccag cttgaagccg
     2761 acgacacacg aggcctcaaa ggagctggac ggttacctgg cctgcgtcag cgatacaagg
     2821 gagtcactgt tgaccttccc tgcaatctgt tcgctgtcga tcaagacaaa cactcccttg
     2881 cctgctagtg cagcgtgtga gagactcttt agcacagcag gactcctgtt cagccctaag
     2941 agagcaaggc ttgataccaa caactttgag aaccagttgc ttctgaaact gaatttacga
     3001 ttttacaact tcgagtgatc tagaactata gtgagtcgta ttacgtagat ccagacatga
     3061 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
     3121 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
     3181 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt
     3241 tttaattcgc ggccgcggcg ccaatgcatt gggcccggta cccagctttt gttcccttta
     3301 gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     3361 ttatccgctc acaattccac acaacatacg agccgggagc ataaagtgta aagcctgggg
     3421 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3481 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3541 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3601 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3661 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3721 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3781 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3841 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3901 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3961 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4021 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4081 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4141 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4201 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4261 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4321 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4381 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4441 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4501 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4561 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4621 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4681 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4741 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4801 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4861 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     4921 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     4981 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5041 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5101 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5161 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5221 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5281 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5341 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5401 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5461 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
     5521 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     5581 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     5641 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     5701 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     5761 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     5821 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     5881 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     5941 tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     6001 ctattacgcc agtcgacc
//
