LOCUS       sequence-255018-        6071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tol2 doxycycline inducible expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6071)
  TITLE     Unpublished
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6071)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAK_Tol2_TRE_Blast
FEATURES             Location/Qualifiers
     source          1..6071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..315
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    9..27
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    45..63
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    80..98
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    116..134
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    152..170
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    187..205
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    223..241
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     288..312
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(400..417)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    406..630
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    680..801
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(691..710)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     745..764
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(910..918,919..954,955..957,958..993,
                     994..996,997..1032,1033..1038,1039..1656))
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             complement(1657..1710)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(1711..2106)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(2116..2626)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2179..2197)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    join(2650..2812,2813..2824)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     promoter        complement(2900..2918)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2901..2918)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2936..2952)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2936..2952)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2949..2971)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2960..2976
                     /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(2984..2990,2991..3008,3009..3014))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3029..3050
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3167..3184)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3338..3926)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3418..3437)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4097..4888,4889..4957))
                     /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     4720..4739
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4958..5062)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5140..5595)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5277..5298)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5489..5508
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5721..5743
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5735..5752
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5736..5752
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5759..5778
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5759..5777
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5790..5801,5802..5989)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 gagtttactc cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag
       61 agaacgatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct
      121 atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg
      181 agtttatccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag
      241 aacgtatgtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg
      301 aaccgtcaga tcgcctggag aattggctag ctagacagga tccacgcgtg gccgcgcgca
      361 ctagtgaatt ccatggatat ctttaaaccc gctgatcagc ctcgactgtg ccttctagtt
      421 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
      481 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
      541 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
      601 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
      661 ctagggggta atcaagcttt aagatacatt gatgagtttg gacaaaccac aactagaatg
      721 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
      781 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
      841 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat
      901 tatgatcagt tacccgggga gcatgtcaag gtcaaaatcg tcaagagcgt cagcaggcag
      961 catatcaagg tcaaagtcgt caagggcatc ggctgggagc atgtctaagt caaaatcgtc
     1021 aagggcgtcg gtcggcccgc cgctttcgca ctttagctgt ttctccaggc cacatatgat
     1081 tagttccagg ccgaaaagga aggcaggttc ggctccctgc cggtcgaaca gctcaattgc
     1141 ttgtctcaga agtgggggca tagaatcggt ggtaggtgtc tctctttcct cttttgctac
     1201 ttgatgctcc tgttcctcca atacgcagcc cagtgtaaag tggcccacgg cggacagagc
     1261 gtacagtgcg ttctccaggg agaagccttg ctgacacagg aacgcgagct gattttccag
     1321 ggtttcgtac tgtttctctg ttgggcgggt gccgagatgc actttagccc cgtcgcgatg
     1381 tgagaggaga gcacagcggt atgacttggc gttgttccgc agaaagtctt gccatgactc
     1441 gccttccagg gggcagaagt gggtatgatg cctgtccagc atctcgattg gcagggcatc
     1501 gagcagggcc cgcttgttct tcacgtgcca gtacagggta ggctgctcaa ctcccagctt
     1561 ttgagcgagt ttccttgtcg tcaggccttc gataccgaca ccattgagta attccagagc
     1621 tccgtttatg actttgctct tgtccagtct agacattgga ccagggtttt cttcaacatc
     1681 accacaagtg aggagagaac ctctaccttc gccctcccac acataaccag agggcagcaa
     1741 ttcacgaatc ccaactgccg tcggctgtcc atcactgtcc ttcactatgg ctttgatccc
     1801 aggatgcaga tcgagaagca cctgtcggca ccgtccgcag gggctcaaga tgcccctgtt
     1861 ctcatttccg atcgcgacga tacaagtcag gttgccagct gccgcagcag cagcagtgcc
     1921 cagcaccacg agttctgcac aaggtccccc agtaaaatga tatacattga caccagtgaa
     1981 gatgcggccg tcgctagaga gagctgcgct ggcgacgctg tagtcttcag agatggggat
     2041 gctgttgatt gtagccgttg ctctttcaat gagggtggat tcttcttgag acaaaggctt
     2101 ggccatggtg aattgctggg gagagaggtc ggtgattcgg tcaacgaggg agccgactgc
     2161 cgacgtgcgc tccggaggct tgcagaatgc ggaacaccgc gcgggcagga acagggccca
     2221 cactaccgcc ccacaccccg cctcccgcac cgccccttcc cggccgctgc tctcggcgcg
     2281 ccctgctgag cagccgctat tggccacagc ccatcgcggt cggcgcgctg ccattgctcc
     2341 ctggcgctgt ccgtctgcga gggtactagt gagacgtgcg gcttccgttt gtcacgtccg
     2401 gcacgccgcg aaccgcaagg aaccttcccg acttaggggc ggagcaggaa gcgtcgccgg
     2461 ggggcccaca agggtagcgg cgaagatccg ggtgacgctg cgaacggacg tgaagaatgt
     2521 gcgagaccca gggtcggcgc cgctgcgttt cccggaacca cgcccagagc agccgcgtcc
     2581 ctgcgcaaac ccagggctgc cttggaaaag gcgcaacccc aacccctaaa caagaatctc
     2641 tagttttctt tcttgctttt acttttactt ccttaatact caagtacaat tttaatggag
     2701 tactttttta cttttactca agtaagattc tagccagata cttttacttt taattgagta
     2761 aaattttccc taagtacttg tactttcact tgagtaaaat ttttgagtac tttttacacc
     2821 tctgctcgac catatgggag agctcccaac gcgatagata tgataattgg aatgcgcgtt
     2881 ggatgcatag cttgagtatt ctatagtgtc acctaaatag cttggcgtaa tcatggtcat
     2941 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     3001 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     3061 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3121 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     3181 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3241 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3301 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3361 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3421 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3481 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3541 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3601 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3661 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3721 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3781 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3841 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3901 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3961 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4021 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4081 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4141 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4201 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4261 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4321 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4381 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     4441 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4501 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4561 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4621 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4681 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4741 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4801 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4861 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4921 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4981 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5041 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa
     5101 ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc gttaatattt
     5161 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     5221 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     5281 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     5341 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     5401 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     5461 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     5521 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     5581 cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
     5641 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag
     5701 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgta
     5761 atacgactca ctatagggcg aattgggccc agaggtgtaa agtacttgag taattttact
     5821 tgattactgt acttaagtat tatttttggg gatttttact ttacttgagt acaattaaaa
     5881 atcaatactt ttacttttac ttaattacat ttttttagaa aaaaaagtac tttttactcc
     5941 ttacaatttt atttacagtc aaaaagtact tattttttgg agatcacttc attctatttt
     6001 cccttgctat taccaaacca attgaattgc gctgatgccc agtttaattt aaatagatct
     6061 atcgatatgt c
//
