LOCUS       sequence-274127-        5803 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for in vitro transcription of Tol2 nls-transposase-nls for 
            zebrafish transgenesis using T3..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5803)
  AUTHORS   Hall TE, Martel N, Ariotti N, Xiong Z, Lo HP, Ferguson C, Rae J, Lim
            YW, Parton RG
  TITLE     In vivo cell biological screening identifies an endocytic capture 
            mechanism for T-tubule formation.
  JOURNAL   Nat Commun. 2020 Jul 24;11(1):3711. doi: 10.1038/s41467-020-17486-w.
  PUBMED    32709891
REFERENCE   2  (bases 1 to 5803)
  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: "Nat 
            Commun."; date: "2020-07-24"; volume: "11(1):3711. doi"; pages: " 
            10.1038/s41467-020-17486-w"
COMMENT     Sequence Label: nls-transposase-nls-pA in pT3TS-Dest
FEATURES             Location/Qualifiers
     source          1..5803
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..69,70..861)
                     /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     complement(219..238)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1032..1620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1521..1540
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1774..1791
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1908..1929
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1944..1949,1950..1967,1968..1974)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1982..1998
                     /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)."
     primer_bind     1987..2009
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2006..2022
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2006..2022
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2043..2061
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     5'UTR           2086..2128
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     protein_bind    2135..2159
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2160..2169
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2178..2198
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2199..4142
                     /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             4143..4163
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4182..4303
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     3'UTR           4377..4502
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     protein_bind    complement(4555..4579)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    4581..4715
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(4778..4798)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     3'UTR           4823..4948
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(5034..5053)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5035..5053)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5060..5077)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5060..5076)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5069..5091)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(5217..5672)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5354..5375)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5566..5585
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5699..5803
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
       61 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
      121 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
      181 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
      241 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
      301 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
      361 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
      421 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
      481 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
      541 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
      601 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
      661 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
      721 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
      781 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
      841 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
      901 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
      961 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     1021 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     1081 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     1141 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     1201 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     1261 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     1321 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     1381 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     1441 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     1501 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     1561 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     1621 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     1681 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     1741 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     1801 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     1861 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     1921 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     1981 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc
     2041 gaaattaacc ctcactaaag ggaacaaaag ctggagctaa gcttgcttgt tctttttgca
     2101 gaagctcaga ataaacgctc aactttggca gatctcaagt ttgtacaaaa aagcaggctg
     2161 ccaccatggc ttctccacct aagaagaaga gaaaggtgga agaggtgtgc gactcttccg
     2221 ccgcagcttc atctaccgtc cagaaccagc ctcaggacca agagcacccc tggccatatc
     2281 tgagagagtt cttctccctg agcggagtga ataaggactc atttaaaatg aagtgtgtgc
     2341 tgtgtcttcc actcaacaaa gagattagcg cgtttaaatc ctcaccctcg aacctgagga
     2401 agcacattga gcgtatgcac cctaactacc tgaaaaatta ctccaagctg accgcccaga
     2461 agagaaagat aggcactagt actcacgcat ccagcagtaa gcagttgaaa gtagactcag
     2521 tgttccctgt taagcatgtg tcccctgtaa cagttaacaa ggctattctc cggtacatta
     2581 tccaaggtct gcacccgttc agcaccgtcg acctaccctc cttcaaggag ctgattagta
     2641 cgcttcagcc tggaattagt gtgattactc gccctactct gagaagcaaa atcgcagaag
     2701 cggcgctgat catgaaacag aaagtgactg ctgcaatgag cgaggtggaa tggatcgcga
     2761 cgacaaccga ctgttggaca gcacgtagga agtcctttat aggcgtcact gcccactgga
     2821 ttaaccccgg ttcactggag cgacactcag cagccctggc ctgcaaacgc cttatgggat
     2881 ctcacacttt tgaggtgtta gcgtctgcta tgaacgacat ccactcggaa tacgaaatta
     2941 gggacaaggt ggtctgtacc acaacagact ctggatctaa ctttatgaaa gcgtttcgcg
     3001 tttttggcgt ggagaacaat gatatcgaaa cggaggccag acgctgtgag tcagacgaca
     3061 ctgacagcga aggctgcggc gaaggttcag acggcgtcga atttcaggac gcgagtcgcg
     3121 ttctcgacca ggatgatgga tttgaatttc agttaccaaa gcaccagaag tgtgcctgtc
     3181 atcttctaaa cctcgtctct tcggtcgacg cacaaaaagc tctgtccaac gagcattaca
     3241 agaagctcta ccggtctgtc ttcggcaagt gtcaggccct ttggaacaag tcctccagat
     3301 ccgctctcgc cgcagaggcg gtagagagtg agtccagact tcagctgctg agacctaatc
     3361 agacacgctg gaacagcact ttcatggcag tggaccgaat cttacaaatc tgtaaagaag
     3421 ccggtgaggg cgctctccgc aacatctgta catcactgga agtccctatg tttaacccag
     3481 ctgagatgct cttcttgaca gagtgggcta acactatgcg gccagtcgct aaagtcctcg
     3541 acatccttca ggccgaaacc aatacacagt taggctggct ccttccaagc gtgcaccaac
     3601 tcagtctgaa gctccaaaga ctgcatcata gtctgcggta ctgcgatccc ctcgtggatg
     3661 ctctgcagca gggaatccag actcggttca aacacatgtt cgaagaccct gagattatcg
     3721 ctgcagccat ccttctccca aaatttcgaa cctcgtggac aaacgatgag accattatta
     3781 agcgcggcat ggattatatc cgcgtccacc ttgaacccct ggaccacaaa aaagagctgg
     3841 ctaattcaag tagtgacgat gaggatttct ttgccagctt gaagccgacg acacacgagg
     3901 cctcaaagga gctggacggt tacctggcct gcgtcagcga tacaagggag tcactgttga
     3961 ccttccctgc aatctgttcg ctgtcgatca agacaaacac tcccttgcct gctagtgcag
     4021 cgtgtgagag actctttagc acagcaggac tcctgttcag ccctaagaga gcaaggcttg
     4081 ataccaacaa ctttgagaac cagttgcttc tgaaactgaa tttacgattt tacaacttcg
     4141 agcctaagaa gaagagaaag gtgtaaggat cccagacatg ataagataca ttgatgagtt
     4201 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     4261 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     4321 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttcggtta ccactaaacc
     4381 agcctcaaga acacccgaat ggagtctcta agctacataa taccaactta cactttacaa
     4441 aatgttgtcc cccaaaatgt agccattcgt atctgctcct aataaaaaga aagtttcttc
     4501 acattctaaa aaaaaaaaaa aaaaaaaaaa aaaaaacccc cccccccccc gtcgacccag
     4561 ctttcttgta caaagtgggg gatccagaca tgataagata cattgatgag tttggacaaa
     4621 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     4681 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     4741 tgtttcaggt tcagggggag gtgtgggagg ttttttccaa ctttattata catagttgct
     4801 cgagcggatc tggttaccac taaaccagcc tcaagaacac ccgaatggag tctctaagct
     4861 acataatacc aacttacact ttacaaaatg ttgtccccca aaatgtagcc attcgtatct
     4921 gctcctaata aaaagaaagt ttcttcacat tctaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4981 aacccccccc cccccctgca gggccaagtc ggcctctaga ggtacccaat tcgccctata
     5041 gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     5101 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     5161 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggaaat
     5221 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     5281 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     5341 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     5401 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     5461 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     5521 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     5581 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     5641 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     5701 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5761 ataaccctga taaatgcttc aataatattg aaaaaggaag agt
//
