LOCUS       sequence-135631-        7847 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  promoterless Tol2 vector with attR1/attR2 cassette and late SV40 
            polyadenylation signal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7847)
  AUTHORS   Villefranc JA, Amigo J, Lawson ND
  TITLE     Gateway compatible vectors for analysis of gene function in the 
            zebrafish.
  JOURNAL   Dev Dyn. 2007 Nov . 236(11):3077-87.
  PUBMED    17948311
REFERENCE   2  (bases 1 to 7847)
  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: "Dev Dyn. 
            2007 Nov . 236(11):3077-87."
COMMENT     Sequence Label: 251 pTol2Dest
FEATURES             Location/Qualifiers
     source          1..7847
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(34..462)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     356..375
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     589..611
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     603..620
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     604..620
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        630..648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(778..789,790..952)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    1353..1477
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1502..1507,1508..1525,1526..1532)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1586..2245
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1652..1671)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2587..2892
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2771..2790
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2933..3057)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    3102..3236
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3126..3145)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3180..3199
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(5377..5564,5565..5576)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5610..5626)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5610..5626)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(5663..5683)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5663..5681)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5702..5718)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5702..5718)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5715..5737)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5726..5742
                     /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(5750..5756,5757..5774,5775..5780))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5795..5816
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5933..5950)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6104..6692)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6184..6203)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6863..7654,7655..7723))
                     /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     7486..7505
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7724..7828)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
      241 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
      301 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
      361 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
      421 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
      481 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
      541 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
      601 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta
      661 caggtcattg cacaacacca gaaatgccct ctgatctgca aaagacgtga atatctgttc
      721 agacacccat atccactctg ttccacacag gtcagaggtt tgtccaggag ttcttgacag
      781 aggtgtaaaa agtactcaaa aattttactc aagtgaaagt acaagtactt agggaaaatt
      841 ttactcaatt aaaagtaaaa gtatctggct agaatcttac ttgagtaaaa gtaaaaaagt
      901 actccattaa aattgtactt gagtattaag gaagtaaaag taaaagcaag aaagaaaact
      961 agagattctt gtttaagctt ttaatctcaa aaaacattaa atgaaatgca tacaaggttt
     1021 tatcctgctt tagaactgtt tgtatttaat tatcaaacta taagacagac aatctaatgc
     1081 cagtacacgc tactcaaagt tgtaaaacct cagatttaac ttcagtagaa gctgattctc
     1141 aaaattgtta gtgtcaagcc tagctctttt ggggctgaaa agcaatcctg cagtgctgaa
     1201 aagcctctca caggcagccg atgcgggaag aggtgtatta gtcttgatag agaggctgca
     1261 aatagcagga aacgtgagca gagactccct ggtgtctgaa acacaggcca gatgggccga
     1321 tatcgctagc atcgattcga attcaaggat caacaagttt gtacaaaaaa gctgaacgag
     1381 aaacgtaaaa tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta
     1441 cataatactg taaaacacaa catatccagt catattggcg gccgcattag gcaccccagg
     1501 ctttacactt tatgcttccg gctcgtataa tgtgtggatt ttgagttagg atccgtcgag
     1561 attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga
     1621 tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac
     1681 ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa
     1741 gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga
     1801 attccgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta
     1861 caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga
     1921 tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc
     1981 ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag
     2041 tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac
     2101 catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca
     2161 tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg
     2221 cgatgagtgg cagggcgggg cgtaaagatc tggatccggc ttactaaaag ccagataaca
     2281 gtatgcgtat ttgcgcgctg atttttgcgg tataagaata tatactgata tgtatacccg
     2341 aagtatgtca aaaagaggta tgctatgaag cagcgtatta cagtgacagt tgacagcgac
     2401 agctatcagt tgctcaaggc atatatgatg tcaatatctc cggtctggta agcacaacca
     2461 tgcagaatga agcccgtcgt ctgcgtgccg aacgctggaa agcggaaaat caggaaggga
     2521 tggctgaggt cgcccggttt attgaaatga acggctcttt tgctgacgag aacaggggct
     2581 ggtgaaatgc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg
     2641 gatgtacaga gtgatattat tgacacgccc gggcgacgga tggtgatccc cctggccagt
     2701 gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat
     2761 gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa
     2821 gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc
     2881 tggggaatat aaatgtcagg ctcccttata cacagccagt ctgcaggtcg accatagtga
     2941 ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa tctaatttaa
     3001 tatattgata tttatatcat tttacgtttc tcgttcagct ttcttgtaca aagtggttga
     3061 tcctctcgag cctctagaac tatagtgagt cgtattacgt agatccagac atgataagat
     3121 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     3181 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     3241 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaat
     3301 tcgcggccgc ggcgccaatg cattgggccc tcgagcatat gagatctgtc ccgatctttc
     3361 tcttctgtgc tgtcaattta gagtagtttt tgaggtaatt tgggtgcatt ctctgtaaaa
     3421 acaacaaata aatatttata ttgtatgcaa taaatgtaac aattacacat acactctctg
     3481 attacagcat taaagggtta gttcacccaa aaatgaaaag atgtcattaa tgactcaccc
     3541 tcatgtcgtt ccaagcccgt aacacctccg ttcatcttcg gaacacagtt taagatattt
     3601 tagatttagt ccgagagctt tctgtccctc cactgaaaat gtatgtacgg tatactgtcc
     3661 atgtccagaa aggtaataaa aacatcaaag tagtccatgt gacatcagtg ggttaggtag
     3721 aattatttga agcatcgaaa atacattttg gtccaaaaat aacaaaacct acgactttat
     3781 tcagcattgt agcgtcactg cgaagtcgtg aacgcggatt gacaacagac ccggaagaga
     3841 atacaatgcc gaataaagtc gtaggttttg ttatttttgg accaaaatgt attcgatgct
     3901 tcaaaaaatt ctaactaacc cactgatgtc acatggacta ctttgatgtt tttattacct
     3961 ttctggacat ggacagtata ccgtacatac attctcaatg gaggcacaga aagctctcgg
     4021 actaaatcta aaatatctta aactgtgttc tgaagatgaa cggaggtctt acgggcttgg
     4081 aacgacatga gggcgagtca ttaatgacat tattttcatt tttgggtgaa ctaacccttt
     4141 aataaaactg acatttgcag ccaattgcac actttataat gttaaatact tgttgatttt
     4201 agaaatacag tagcatttag tgacaatcaa acattaattt tatttctgat tatattgaca
     4261 aaaacacagg actgtaaggc tgacatgaca gggtgaattg atttcattac attagttttg
     4321 atggtaacac ttttacaata aggttcgtta gttaactaca ttagttaaca tgaactaata
     4381 atgaactgca cgtatacatg ttaatttcaa catttactaa tactttatat cttgttaaca
     4441 ttagttaatg cactgtgaac taacatgaac aaacaatgaa catctgtatt tctattaact
     4501 aacgttaaca aagattaaca gatacagtaa caaatgtatt gctcatggtt atttaatgtt
     4561 agttaataca tgaactaatc aacctcattg taaagtgtta ccgttttgac aatacaacat
     4621 gtttggaaaa aaatttctcg cttggttttc ttgtgttttg catgaattca aacacaatga
     4681 gagcacggaa tgataatgat tattcaaagc tgcaataggc gccaaatttc ccgtggacct
     4741 aacgttacca attacaaatg cattttagca ttttattacg tgagccttat attgtcaatc
     4801 gcatgcatta gtgaaaatag gcctactaca catataaaag gcgcgccatc aacgtcaaaa
     4861 catgcacacc caaaaaaaaa aaaaaaaaaa aaaaaaaaaa ctatcagtaa aacaaaatac
     4921 ttaatgtact tacctcaata tgcttcctta ggtttgatgg cgaacttttg aaggccgata
     4981 tttctttatt aagcgggaga cagaggacac atttcatctt gaatgaatct ttatttacac
     5041 cacttaaaga aaagaattcg cgaagatacg gccacgggtg ctcttgatcc tgtggctgat
     5101 tttggactgt gctgctcgca gctgctgatg aatcacatac ttcctccatt ttcttccact
     5161 gattgactgt tataatttcc ctaatttcca ggtcaaggtg ctgtgcattg tggtaataga
     5221 tgtgacatga cgtcacttcc aaaggaccaa tgaacatgtc tgaccaattt catataatgt
     5281 gaaaacgatt ttcataggca gaataaataa catttaaatt aaactgggca tcagcgcaat
     5341 tcaattggtt tggtaatagc aagggaaaat agaatgaagt gatctccaaa aaataagtac
     5401 tttttgactg taaataaaat tgtaaggagt aaaaagtact tttttttcta aaaaaatgta
     5461 attaagtaaa agtaaaagta ttgattttta attgtactca agtaaagtaa aaatccccaa
     5521 aaataatact taagtacagt aatcaagtaa aattactcaa gtactttaca cctctggttc
     5581 ttgaccccct accttcagca agcccagcag atccactagt tctagagcgg ccgccaccgc
     5641 ggtggagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     5701 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     5761 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     5821 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5881 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     5941 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     6001 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6061 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6121 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6181 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6241 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6301 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6361 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6421 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6481 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6541 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6601 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6661 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6721 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6781 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6841 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6901 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6961 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     7021 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7081 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7141 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7201 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7261 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7321 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7381 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7441 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7501 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7561 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7621 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7681 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7741 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7801 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtg
//
