LOCUS       sequence-224315-        7408 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  functional recombinant Tol2 protein from E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7408)
  AUTHORS   Ni J, Wangensteen KJ, Nelsen D, Balciunas D, Skuster KJ, Urban MD, 
            Ekker SC
  TITLE     Active recombinant Tol2 transposase for gene transfer and gene 
            discovery applications.
  JOURNAL   Mob DNA. 2016 Mar 31;7:6. doi: 10.1186/s13100-016-0062-z. 
            eCollection 2016.
  PUBMED    27042235
REFERENCE   2  (bases 1 to 7408)
  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: "Mob DNA."; 
            date: "2016-03-31"; volume: "7:6. doi"; pages: " 
            10.1186/s13100-016-0062-z. eCollection 2016"
COMMENT     Sequence Label: pET21-HIS-Tol2
FEATURES             Location/Qualifiers
     source          1..7408
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      27..482
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(114..133)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     324..345
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        508..612
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(613..681,682..1473)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(831..850)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1644..2232
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2133..2152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2386..2403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2418..2557
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2483..2502)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2643..2665
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2659..2850)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3659..4741)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4703..4722
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4742..4819)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5025..5044
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5128..5147
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5128..5146
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5147..5171
                     /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)."
     RBS             5186..5208
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5219..5236
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5237..7183
                     /codon_start=1
                     /product="transposase of the Tol2 transposon from the 
                     medaka fish Oryzias latipes (Kawakami, 2007)"
                     /label=Tol2 transposase
                     /translation="EEVCDSSAAASSTVQNQPQDQEHPWPYLREFFSLSGVNKDSFKMK
                     CVLCLPLNKEISAFKSSPSNLRKHIERMHPNYLKNYSKLTAQKRKIGTSTHASSSKQLK
                     VDSVFPVKHVSPVTVNKAILRYIIQGLHPFSTVDLPSFKELISTLQPGISVITRPTLRS
                     KIAEAALIMKQKVTAAMSEVEWIATTTDCWTARRKSFIGVTAHWINPGSLERHSAALAC
                     KRLMGSHTFEVLASAMNDIHSEYEIRDKVVCTTTDSGSNFMKAFRVFGVENNDIETEAR
                     RCESDDTDSEGCGEGSDGVEFQDASRVLDQDDGFEFQLPKHQKCACHLLNLVSSVDAQK
                     ALSNEHYKKLYRSVFGKCQALWNKSSRSALAAEAVESESRLQLLRPNQTRWNSTFMAVD
                     RILQICKEAGEGALRNICTSLEVPMFNPAEMLFLTEWANTMRPVAKVLDILQAETNTQL
                     GWLLPSVHQLSLKLQRLHHSLRYCDPLVDALQQGIQTRFKHMFEDPEIIAAAILLPKFR
                     TSWTNDETIIKRGMDYIRVHLEPLDHKKELANSSSDDEDFFASLKPTTHEASKELDGYL
                     ACVSDTRESLLTFPAICSLSIKTNTPLPASAACERLFSTAGLLFSPKRARLDTNNFENQ
                     LLLKLNLRFYNFE"
     CDS             7267..7284
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7337..7355)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7351..7398
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
       61 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
      121 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
      181 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
      241 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
      301 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
      361 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
      421 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
      481 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
      541 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
      601 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
      661 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
      721 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
      781 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
      841 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
      901 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
      961 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     1021 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     1081 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     1141 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     1201 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     1261 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     1321 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     1381 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     1441 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     1501 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     1561 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     1621 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     1681 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     1741 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     1801 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     1861 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     1921 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     1981 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     2041 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     2101 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     2161 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     2221 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     2281 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     2341 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     2401 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     2461 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     2521 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     2581 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     2641 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     2701 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     2761 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     2821 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     2881 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     2941 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     3001 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     3061 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     3121 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     3181 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     3241 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     3301 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     3361 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     3421 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     3481 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     3541 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     3601 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     3661 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3721 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     3781 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     3841 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     3901 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     3961 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     4021 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     4081 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     4141 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     4201 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     4261 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     4321 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     4381 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     4441 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     4501 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     4561 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     4621 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     4681 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     4741 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     4801 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     4861 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     4921 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     4981 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     5041 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     5101 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     5161 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgca
     5221 tcatcatcat catcatgagg aagtatgtga ttcatcagca gctgcgagca gcacagtcca
     5281 aaatcagcca caggatcaag agcacccgtg gccgtatctt cgcgaattct tttctttaag
     5341 tggtgtaaat aaagattcat tcaagatgaa atgtgtcctc tgtctcccgc ttaataaaga
     5401 aatatcggcc ttcaaaagtt cgccatcaaa cctaaggaag catattgaga gaatgcaccc
     5461 aaattacctc aaaaactact ctaaattgac agcacagaag agaaagatcg ggacctccac
     5521 ccatgcttcc agcagtaagc aactgaaagt tgactcagtt ttcccagtca aacatgtgtc
     5581 tccagtcact gtgaacaaag ctatattaag gtacatcatt caaggacttc atcctttcag
     5641 cactgttgat ctgccatcat ttaaagagct gattagtaca ctgcagcctg gcatttctgt
     5701 cattacaagg cctactttac gctccaagat agctgaagct gctctgatca tgaaacagaa
     5761 agtgactgct gccatgagtg aagttgaatg gattgcaacc acaacggatt gttggactgc
     5821 acgtagaaag tcattcattg gtgtaactgc tcactggatc aaccctggaa gtcttgaaag
     5881 acattccgct gcacttgcct gcaaaagatt aatgggctct catacttttg aggtactggc
     5941 cagtgccatg aatgatatcc actcagagta tgaaatacgt gacaaggttg tttgcacaac
     6001 cacagacagt ggttccaact ttatgaaggc tttcagagtt tttggtgtgg aaaacaatga
     6061 tatcgagact gaggcaagaa ggtgtgaaag tgatgacact gattctgaag gctgtggtga
     6121 gggaagtgat ggtgtggaat tccaagatgc ctcacgagtc ctggaccaag acgatggctt
     6181 cgaattccag ctaccaaaac atcaaaagtg tgcctgtcac ttacttaacc tagtctcaag
     6241 cgttgatgcc caaaaagctc tctcaaatga gcactacaag aaactctaca gatctgtctt
     6301 tggcaaatgc caagctttat ggaataaaag cagccgatcg gctctagcag ctgaagctgt
     6361 tgaatcagaa agccggcttc agcttttaag gccaaaccaa acgcggtgga attcaacttt
     6421 tatggctgtt gacagaattc ttcaaatttg caaagaagca ggagaaggcg cacttcggaa
     6481 tatatgcacc tctcttgagg ttccaatgtt taatccagca gaaatgctgt tcttgacaga
     6541 gtgggccaac acaatgcgtc cagttgcaaa agtactcgac atcttgcaag cggaaacgaa
     6601 tacacagctg gggtggctgc tgcctagtgt ccatcagtta agcttgaaac ttcagcgact
     6661 ccaccattct ctcaggtact gtgacccact tgtggatgcc ctacaacaag gaatccaaac
     6721 acgattcaag catatgtttg aagatcctga gatcatagca gctgccatcc ttctccctaa
     6781 atttcggacc tcttggacaa atgatgaaac catcataaaa cgaggcatgg actacatcag
     6841 agtgcatctg gagcctttgg accacaagaa ggaattggcc aacagttcat ctgatgatga
     6901 agatttcttc gcttctttga aaccgacaac acatgaagcc agcaaagagt tggatggata
     6961 tctggcctgt gtttcagaca ccagggagtc tctgctcacg tttcctgcta tttgcagcct
     7021 ctctatcaag actaatacac ctcttcccgc atcggctgcc tgtgagaggc ttttcagcac
     7081 tgcaggattg cttttcagcc ccaaaagagc taggcttgac actaacaatt ttgagaatca
     7141 gcttctactg aagttaaatc tgaggtttta caactttgag tagaagggcg aattccagca
     7201 cactggcggc cgttactagt ggatccgaat tcgagctccg tcgacaagct tgcggccgca
     7261 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     7321 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     7381 gtcttgaggg gttttttgct gaaaggag
//
