LOCUS       sequence-320440-        7660 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector with bidirectional promoter controls expression firefly 
            Luciferase with TRE and Tet-off transactivator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7660)
  AUTHORS   Gan SU, Fu Z, Sia KC, Kon OL, Calne R, Lee KO
  TITLE     Development of a liver-specific Tet-off AAV8 vector for improved 
            safety of insulin gene therapy for diabetes.
  JOURNAL   J Gene Med. 2019 Jan;21(1):e3067. doi: 10.1002/jgm.3067. Epub 2019 
            Jan 20.
  PUBMED    30592790
REFERENCE   2  (bases 1 to 7660)
  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: "J Gene 
            Med."; date: "2019-01"; volume: "21(1):e3067. doi"; pages: " 
            10.1002/jgm.3067. Epub 2019 Jan 20"
COMMENT     Sequence Label: pAAV-Tetoffbidir -Alb-luc
FEATURES             Location/Qualifiers
     source          1..7660
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     polyA_signal    183..304
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(729..749)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          879..944
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(1043..2695)
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     2611..2629
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     protein_bind    join(2933..2951,2952..2974,2975..2993,2994..3016,3017..3035,
                     3036..3058,3059..3077,3078..3100,3101..3119,3120..3142,
                     3143..3161,3162..3184,3185..3203)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     CDS             join(3449..4066,4067..4072,4073..4108,4109..4111,4112..4147,
                     4148..4150,4151..4186,4187..4195)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=tTA-Advanced
                     /note="In the Tet-Off(R) system, tTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     polyA_signal    4205..4339
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   4710..4839
                     /label=AAV2 ITR
     primer_bind     complement(4858..4875)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4858..4874)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4867..4889)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5016..5471
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5103..5122)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5313..5334
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5497..5601
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5602..5670,5671..6462)
                     /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(5820..5839)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6633..7221
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7122..7141
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    7509..7530
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7545..7550,7551..7568,7569..7575)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7583..7599
                     /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     7588..7610
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7607..7623
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7607..7623
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgat ctgcagacat
      181 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
      241 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
      301 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
      361 tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc tctagtcaag
      421 gcactataca tcaaatattc cttattaacc cctttacaaa ttaaaaagct aaaggtacac
      481 aatttttgag catagttatt aatagcagac actctatgcc tgtgtggagt aagaaaaaac
      541 agtatgttat gattataact gttatgccta cttataaagg ttacagaata tttttccata
      601 attttcttgt atagcagtgc agctttttcc tttgtggtgt aaatagcaaa gcaagcaaga
      661 gttctattac taaacacagc atgactcaaa aaacttagca attctgaagg aaagtccttg
      721 gggtcttcta cctttctctt cttttttgga ggagtagaat gttgagagtc agcagtagcc
      781 tcatcatcac tagatggcat ttcttctgag caaaacaggt tttcctcatt aaaggcattc
      841 caccactgct cccattcatc agttccatag gttggaatct aaaatacaca aacaattaga
      901 atcagtagtt taacacatta tacacttaaa aattttatat ttaccttaga gctttaaatc
      961 tctgtaggta gtttgtccaa ttatgtcaca ccacagaagt aaggttcctt cacaaagatc
     1021 ctctagctag agtcgcggcc gcttacacgg cgatctttcc gcccttcttg gcctttatga
     1081 ggatctctct gatttttctt gcgtcgagtt ttccggtaag acctttcggt acttcgtcca
     1141 caaacacaac tcctccgcgc aactttttcg cggttgttac ttgactggcg acgtaatcca
     1201 cgatctcttt ttccgtcatc gtctttccgt gctccaaaac aacaacggcg gcgggaagtt
     1261 caccggcgtc atcgtcggga agacctgcga cacctgcgtc gaagatgttg gggtgttgga
     1321 gcaagatgga ttccaattca gcgggagcca cctgatagcc tttgtactta atcagagact
     1381 tcaggcggtc aacgatgaag aagtgttcgt cttcgtccca gtaagctatg tctccagaat
     1441 gtagccatcc atccttgtca atcaaggcgt tggtcgcttc cggattgttt acataaccgg
     1501 acataatcat aggacctctc acacacagtt cgcctctttg attaacgccc agcgttttcc
     1561 cggtatccag atccacaacc ttcgcttcaa aaaatggaac aactttaccg accgcgcccg
     1621 gtttatcatc cccctcgggt gtaatcagaa tagctgatgt agtctcagtg agcccatatc
     1681 cttgcctgat acctggcaga tggaacctct tggcaaccgc ttccccgact tccttagaga
     1741 ggggagcgcc accagaagca atttcgtgta aattagataa atcgtatttg tcaatcagag
     1801 tgcttttggc gaagaaggag aatagggttg gcaccagcag cgcactttga atcttgtaat
     1861 cctgaaggct cctcagaaac agctcttctt caaatctata cattaagacg actcgaaatc
     1921 cacatatcaa atatccgagt gtagtaaaca ttccaaaacc gtgatggaat ggaacaacac
     1981 ttaaaatcgc agtatccgga atgatttgat tgccaaaaat aggatctctg gcatgcgaga
     2041 atctcacgca ggcagttcta tgaggcagag cgacaccttt aggcagacca gtagatccag
     2101 aggagttcat gatcagtgca attgtcttgt ccctatcgaa ggactctggc acaaaatcgt
     2161 attcattaaa accgggaggt agatgagatg tgacgaacgt gtacatcgac tgaaatccct
     2221 ggtaatccgt tttagaatcc atgataataa ttttttggat gattgggagc tttttttgca
     2281 cgttcaaaat tttttgcaac ccctttttgg aaacgaacac cacggtaggc tgcgaaatgc
     2341 ccatactgtt gagcaattca cgttcattat aaatgtcgtt cgcgggcgca actgcaactc
     2401 cgataaataa cgcgcccaac accggcataa agaattgaag agagttttca ctgcatacga
     2461 cgattctgtg atttgtattc agcccatatc gtttcatagc ttctgccaac cgaacggaca
     2521 tttcgaagta ctcagcgtaa gtgatgtcca cctcgatatg tgcatctgta aaagcaattg
     2581 ttccaggaac cagggcgtat ctcttcatag ccttatgcag ttgctctcca gcggttccat
     2641 cttccagcgg atagaatggc gccgggcctt tctttatgtt tttggcgtct tccatgtcga
     2701 cttagtgggg ttgataggaa aggtgatcgt gtgtgcagaa agactcgctc taatatactt
     2761 ctttaaccaa taactgtaga tcattaacca tacttacctc gcatttcatt ggttcctacc
     2821 ccattacaaa atcataccat ctttgccaaa aagttgtttg actaaatccc ttgcgtatgt
     2881 ttgccatctg gagctgtccg cggtcccggg taccgagctc gactttcact tttctctatc
     2941 actgataggg agtggtaaac tcgactttca cttttctcta tcactgatag ggagtggtaa
     3001 actcgacttt cacttttctc tatcactgat agggagtggt aaactcgact ttcacttttc
     3061 tctatcactg atagggagtg gtaaactcga ctttcacttt tctctatcac tgatagggag
     3121 tggtaaactc gactttcact tttctctatc actgataggg agtggtaaac tcgactttca
     3181 cttttctcta tcactgatag ggagtggtaa actcgacgcg taagagctcg gtaccgctag
     3241 cgctacagct ccagatggca aacatacgca agggatttag tcaaacaact ttttggcaaa
     3301 gatggtatga ttttgtaatg gggtaggaac caatgaaatg cgaggtaagt atggttaatg
     3361 atctacagtt attggttaaa gaagtatatt agagcgagtc tttctgcaca cacgatcacc
     3421 tttcctatca accccactaa gaattcatat gtctagactg gacaagagca aagtcataaa
     3481 ctctgctctg gaattactca atgaagtcgg tatcgaaggc ctgacgacaa ggaaactcgc
     3541 tcaaaagctg ggagttgagc agcctaccct gtactggcac gtgaagaaca agcgggccct
     3601 gctcgatgcc ctggcaatcg agatgctgga caggcatcat acccacttct gccccctgga
     3661 aggcgagtca tggcaagact ttctgcggaa caacgccaag tcattccgct gtgctctcct
     3721 ctcacatcgc gacggggcta aagtgcatct cggcacccgc ccaacagaga aacagtacga
     3781 aaccctggaa aatcagctcg cgttcctgtg tcagcaaggc ttctccctgg agaacgcact
     3841 gtacgctctg tccgccgtgg gccactttac actgggctgc gtattggagg atcaggagca
     3901 tcaagtagca aaagaggaaa gagagacacc taccaccgat tctatgcccc cacttctgag
     3961 acaagcaatt gagctgttcg accatcaggg agccgaacct gccttccttt tcggcctgga
     4021 actaatcata tgtggcctgg agaaacagct aaagtgcgaa agcggcgggc cggccgacgc
     4081 ccttgacgat tttgacttag acatgctccc agccgatgcc cttgacgact ttgaccttga
     4141 tatgctgcct gctgacgctc ttgacgattt tgaccttgac atgctccccg ggtaactaag
     4201 taaggatcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     4261 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     4321 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     4381 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta
     4441 tgatcctgca agcctcgtcg tctggccgga ccacgctatc tgtgcaaggt ccccggacgc
     4501 gcgctccatg agcagagcgc ccgccgccga ggcaagactc gggcggcgcc ctgcccgtcc
     4561 caccaggtca acaggcggta accggcctct tcatcgggaa tgcgcgcgac cttcagcatc
     4621 gccggcatgt cccctggcgg acgggaagta tcagctcgac caagctcaga tcgtagataa
     4681 gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc
     4741 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     4801 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc cttaattaac ctaattcact
     4861 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     4921 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     4981 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag
     5041 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     5101 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5161 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5221 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5281 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5341 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     5401 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     5461 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5521 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5581 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5641 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5701 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5761 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5821 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     5881 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5941 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     6001 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     6061 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     6121 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     6181 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6241 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6301 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6361 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6421 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6481 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6541 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6601 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6661 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6721 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6781 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6841 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6901 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6961 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     7021 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     7081 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     7141 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     7201 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     7261 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     7321 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     7381 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     7441 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     7501 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     7561 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     7621 gaccatgatt acgccagatt taattaaggc cttaattagg
//
