LOCUS       sequence-327067-        7674 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 7674)
  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 7674)
  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..7674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     polyA_signal    175..309
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(734..754)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          884..949
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(1048..2700)
                     /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     2616..2634
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     protein_bind    join(2938..2956,2957..2979,2980..2998,2999..3021,3022..3040,
                     3041..3063,3064..3082,3083..3105,3106..3124,3125..3147,
                     3148..3166,3167..3189,3190..3208)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     CDS             join(3452..4069,4070..4075,4076..4111,4112..4114,4115..4150,
                     4151..4153,4154..4189,4190..4198)
                     /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    4208..4342
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   4724..4853
                     /label=AAV2 ITR
     primer_bind     complement(4891..4907)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4891..4907)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4904..4926)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4915..4931
                     /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(4939..4945,4946..4963,4964..4969))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4984..5005
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5293..5881)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5373..5392)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6052..6843,6844..6912))
                     /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     6675..6694
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6913..7017)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7043..7498)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7180..7201)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7392..7411
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7625..7647
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7639..7656
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7640..7656
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgat cttcgatcca
      181 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
      241 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
      301 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg
      361 gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta tgatctctag
      421 tcaaggcact atacatcaaa tattccttat taaccccttt acaaattaaa aagctaaagg
      481 tacacaattt ttgagcatag ttattaatag cagacactct atgcctgtgt ggagtaagaa
      541 aaaacagtat gttatgatta taactgttat gcctacttat aaaggttaca gaatattttt
      601 ccataatttt cttgtatagc agtgcagctt tttcctttgt ggtgtaaata gcaaagcaag
      661 caagagttct attactaaac acagcatgac tcaaaaaact tagcaattct gaaggaaagt
      721 ccttggggtc ttctaccttt ctcttctttt ttggaggagt agaatgttga gagtcagcag
      781 tagcctcatc atcactagat ggcatttctt ctgagcaaaa caggttttcc tcattaaagg
      841 cattccacca ctgctcccat tcatcagttc cataggttgg aatctaaaat acacaaacaa
      901 ttagaatcag tagtttaaca cattatacac ttaaaaattt tatatttacc ttagagcttt
      961 aaatctctgt aggtagtttg tccaattatg tcacaccaca gaagtaaggt tccttcacaa
     1021 agatcctcta gctagagtcg cggccgctta cacggcgatc tttccgccct tcttggcctt
     1081 tatgaggatc tctctgattt ttcttgcgtc gagttttccg gtaagacctt tcggtacttc
     1141 gtccacaaac acaactcctc cgcgcaactt tttcgcggtt gttacttgac tggcgacgta
     1201 atccacgatc tctttttccg tcatcgtctt tccgtgctcc aaaacaacaa cggcggcggg
     1261 aagttcaccg gcgtcatcgt cgggaagacc tgcgacacct gcgtcgaaga tgttggggtg
     1321 ttggagcaag atggattcca attcagcggg agccacctga tagcctttgt acttaatcag
     1381 agacttcagg cggtcaacga tgaagaagtg ttcgtcttcg tcccagtaag ctatgtctcc
     1441 agaatgtagc catccatcct tgtcaatcaa ggcgttggtc gcttccggat tgtttacata
     1501 accggacata atcataggac ctctcacaca cagttcgcct ctttgattaa cgcccagcgt
     1561 tttcccggta tccagatcca caaccttcgc ttcaaaaaat ggaacaactt taccgaccgc
     1621 gcccggttta tcatccccct cgggtgtaat cagaatagct gatgtagtct cagtgagccc
     1681 atatccttgc ctgatacctg gcagatggaa cctcttggca accgcttccc cgacttcctt
     1741 agagagggga gcgccaccag aagcaatttc gtgtaaatta gataaatcgt atttgtcaat
     1801 cagagtgctt ttggcgaaga aggagaatag ggttggcacc agcagcgcac tttgaatctt
     1861 gtaatcctga aggctcctca gaaacagctc ttcttcaaat ctatacatta agacgactcg
     1921 aaatccacat atcaaatatc cgagtgtagt aaacattcca aaaccgtgat ggaatggaac
     1981 aacacttaaa atcgcagtat ccggaatgat ttgattgcca aaaataggat ctctggcatg
     2041 cgagaatctc acgcaggcag ttctatgagg cagagcgaca cctttaggca gaccagtaga
     2101 tccagaggag ttcatgatca gtgcaattgt cttgtcccta tcgaaggact ctggcacaaa
     2161 atcgtattca ttaaaaccgg gaggtagatg agatgtgacg aacgtgtaca tcgactgaaa
     2221 tccctggtaa tccgttttag aatccatgat aataattttt tggatgattg ggagcttttt
     2281 ttgcacgttc aaaatttttt gcaacccctt tttggaaacg aacaccacgg taggctgcga
     2341 aatgcccata ctgttgagca attcacgttc attataaatg tcgttcgcgg gcgcaactgc
     2401 aactccgata aataacgcgc ccaacaccgg cataaagaat tgaagagagt tttcactgca
     2461 tacgacgatt ctgtgatttg tattcagccc atatcgtttc atagcttctg ccaaccgaac
     2521 ggacatttcg aagtactcag cgtaagtgat gtccacctcg atatgtgcat ctgtaaaagc
     2581 aattgttcca ggaaccaggg cgtatctctt catagcctta tgcagttgct ctccagcggt
     2641 tccatcttcc agcggataga atggcgccgg gcctttcttt atgtttttgg cgtcttccat
     2701 gtcgacttag tggggttgat aggaaaggtg atctgtgtgc agaaagactc gctctaatat
     2761 acttctttaa ccaataactg tagatcatta accatactta cctcgcattt cattggttcc
     2821 taccccatta caaaatcata ccatctttgc caaaaagttg tttgactaaa tcccttgcgt
     2881 atgtttgcca tctggagctg tccgcggtcc ccgggtaccg agctcgactt tcacttttct
     2941 ctatcactga tagggagtgg taaactcgac tttcactttt ctctatcact gatagggagt
     3001 ggtaaactcg actttcactt ttctctatca ctgataggga gtggtaaact cgactttcac
     3061 ttttctctat cactgatagg gagtggtaaa ctcgactttc acttttctct atcactgata
     3121 gggagtggta aactcgactt tcacttttct ctatcactga tagggagtgg taaactcgac
     3181 tttcactttt ctctatcact gatagggagt ggtaaactcg agcgtaagag ctcggtaccg
     3241 ctagcgctac agctccagat ggcaaacata cgcaagggat ttagtcaaac aactttttgg
     3301 caaagatggt atgattttgt aatggggtag gaaccaatga aatgcgaggt aagtatggtt
     3361 aatgatctac agttattggt taaagaagta tattagagcg agtctttctg cacacagatc
     3421 acctttccta tcaaccccac taagaattca tatgtctaga ctggacaaga gcaaagtcat
     3481 aaactctgct ctggaattac tcaatgaagt cggtatcgaa ggcctgacga caaggaaact
     3541 cgctcaaaag ctgggagttg agcagcctac cctgtactgg cacgtgaaga acaagcgggc
     3601 cctgctcgat gccctggcaa tcgagatgct ggacaggcat catacccact tctgccccct
     3661 ggaaggcgag tcatggcaag actttctgcg gaacaacgcc aagtcattcc gctgtgctct
     3721 cctctcacat cgcgacgggg ctaaagtgca tctcggcacc cgcccaacag agaaacagta
     3781 cgaaaccctg gaaaatcagc tcgcgttcct gtgtcagcaa ggcttctccc tggagaacgc
     3841 actgtacgct ctgtccgccg tgggccactt tacactgggc tgcgtattgg aggatcagga
     3901 gcatcaagta gcaaaagagg aaagagagac acctaccacc gattctatgc ccccacttct
     3961 gagacaagca attgagctgt tcgaccatca gggagccgaa cctgccttcc ttttcggcct
     4021 ggaactaatc atatgtggcc tggagaaaca gctaaagtgc gaaagcggcg ggccggccga
     4081 cgcccttgac gattttgact tagacatgct cccagccgat gcccttgacg actttgacct
     4141 tgatatgctg cctgctgacg ctcttgacga ttttgacctt gacatgctcc ccgggtaact
     4201 aagtaaggat ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     4261 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     4321 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     4381 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga
     4441 ttatgatcct gcaagcctcg tcgtcctggc cggaccacgc tatctgtgca aggtccccgg
     4501 ccccggacgc gcgctccatg agcagagcgc ccgccgccga ggcgaagact cgggcggcgc
     4561 cctgcccgtc ccaccaggtc aacaggcggt aaccggcctc ttcatcggga atgcgcgcga
     4621 ccttcagcat cgccggcatg tccccctggc ggacgggaag tatccagctc gaccaagctg
     4681 aagatcgtag ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg
     4741 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     4801 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagcctaatt
     4861 aaggccttaa ttaaatctgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4921 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4981 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5041 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5101 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5161 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5221 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5281 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5341 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5401 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5461 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5521 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5581 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5641 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5701 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5761 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5821 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5881 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5941 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6001 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6061 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6121 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6181 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6241 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6301 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6361 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6421 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6481 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6541 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6601 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6661 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6721 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6781 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6841 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6901 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6961 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7021 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     7081 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     7141 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     7201 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     7261 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     7321 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     7381 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     7441 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     7501 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     7561 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     7621 ttttcccagt cacgacgttg taaaacgacg gccagtgaat taggttaatt aagg
//
