LOCUS       Exported                6565 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV vector, pTet bidi (bidirectional tetracycline-responsive 
            promoter) driving Tet-dependent rtTA and eGFP expression.
ACCESSION   .
VERSION     .
KEYWORDS    pAC1-V16-eGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6565)
  AUTHORS   Chtarto A, Humbert-Claude M, Bockstael O, Das AT, Boutry S, Breger 
            LS, Klaver B, Melas C, Barroso-Chinea P, Gonzalez-Hernandez T, 
            Muller RN, DeWitte O, Levivier M, Lundberg C, Berkhout B, Tenenbaum 
            L
  TITLE     A regulatable AAV vector mediating GDNF biological effects at 
            clinically-approved sub-antimicrobial doxycycline doses.
  JOURNAL   Mol Ther Methods Clin Dev. 2016 Mar 30;5:16027. doi: 
            10.1038/mtm.2016.27. eCollection 2016.
  PUBMED    27069954
REFERENCE   2  (bases 1 to 6565)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6565
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    1..271
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     protein_bind    43..313
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     regulatory      485..494
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             491..1210
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             491..1207
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(536..557)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(797..816)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1144..1165
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          1310..1375
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1505..1525
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1950..2084
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2201..2218)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2372..2960)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2452..2471)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3131..3991)
                     /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     3754..3773
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3992..4096)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4164..4182
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4220..4242)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4342..4361
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4428..4883)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4565..4586)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4777..4796
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5027..5156
                     /label=AAV2 ITR
     polyA_signal    5503..5637
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5647..6393)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKIINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     promoter        complement(6495..6533)
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 tctctatcac tgatagggag tggtaaactc gactttcact tttctctatc actgataggg
       61 agtggtaaac tcgactttca cttttctcta tcactgatag ggagtggtaa actcgacttt
      121 cacttttctc tatcactgat agggagtggt aaactcgact ttcacttttc tctatcactg
      181 atagggagtg gtaaactcga ctttcacttt tctctatcac tgatagggag tggtaaactc
      241 gactttcact tttctctatc actgataggg agtggtaaac tcgactttca cttttctcta
      301 tcactgatag ggagtggtaa actcgaccta tataagcaga gctcgtttag tgaaccgtca
      361 gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc
      421 cagcctccgc ggccccgaat tctgcagtcg acggtaccgc gggcccggga tcgatccacc
      481 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
      541 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
      601 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
      661 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
      721 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
      781 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
      841 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
      901 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
      961 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     1021 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1081 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     1141 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     1201 gtacaagtaa agcggccgcg actctagcta gaggatcttt gtgaaggaac cttacttctg
     1261 tggtgtgaca taattggaca aactacctac agagatttaa agctctaagg taaatataaa
     1321 atttttaagt gtataatgtg ttaaactact gattctaatt gtttgtgtat tttagattcc
     1381 aacctatgga actgatgaat gggagcagtg gtggaatgcc tttaatgagg aaaacctgtt
     1441 ttgctcagaa gaaatgccat ctagtgatga tgaggctact gctgactctc aacattctac
     1501 tcctccaaaa aagaagagaa aggtagaaga ccccaaggac tttccttcag aattgctaag
     1561 ttttttgagt catgctgtgt ttagtaatag aactcttgct tgctttgcta tttacaccac
     1621 aaaggaaaaa gctgcactgc tatacaagaa aattatggaa aaatattctg taacctttat
     1681 aagtaggcat aacagttata atcataacat actgtttttt cttactccac acaggcatag
     1741 agtgtctgct attaataact atgctcaaaa attgtgtacc tttagctttt taatttgtaa
     1801 aggggttaat aaggaatatt tgatgtatag tgccttgact agagatcata atcagccata
     1861 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     1921 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     1981 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     2041 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcgaagat ctgaggaacc
     2101 cctagtgatg gagttggcca ctccctctct gcagcctgca ttaatgaatc ggccaacgcg
     2161 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2221 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2281 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2341 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2401 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2461 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2521 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2581 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2641 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2701 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2761 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2821 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2881 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2941 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3001 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3061 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3121 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3181 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3241 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3301 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3361 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3421 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3481 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3541 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3601 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3661 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3721 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3781 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3841 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3901 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     3961 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4021 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4081 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     4141 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     4201 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     4261 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     4321 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
     4381 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt
     4441 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     4501 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     4561 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     4621 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     4681 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     4741 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     4801 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     4861 taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg
     4921 cgatcggtgc gggcctcttc gctattacgc caggctgcag gggggggggg ggggggsccc
     4981 mcysmskggg gggggggggg gggggggggg gggggccact ccctctctgc gcgctcgctc
     5041 gctcactgag gccgcccggg caaagcccgg gcgtcgggcg acctttggtc gcccggcctc
     5101 agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctcaga
     5161 tcttcagctt ggtcgagctg gatacttccc gtccgccagg gggacatgcc ggcgatgctg
     5221 aaggtcgcgc gcattcccga tgaagaggcc ggttaccgcc tgttgacctg gtgggacggg
     5281 cagggcgccg cccgagtctt cgcctcggcg gcgggcgctc tgctcatgga gcgcgcgtcc
     5341 ggggccgggg accttgcaca gatagcgtgg tccggccagg acgacgaggc ttgcaggatc
     5401 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     5461 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     5521 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5581 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcctt
     5641 acttagttac ccggggagca tgtcaaggtc aaaatcgtca agagcgtcag caggcagcat
     5701 atcaaggtca aagtcgtcaa gggcatcggc tgggagcatg tctaagtcaa aatcgtcaag
     5761 ggcgtcggtc ggcccgccgc tttcgcactt tagctgtttc tccaggccac atatgattag
     5821 ttccaggccg aaaaggaagg caggttcggc tccctgccgg tcgaacagct caattgcttg
     5881 tttcagaagt gggggcatag aatcggtggt aggtgtctct ctttcctctt ttgctacttg
     5941 atgctcctgt tcctccaata cgcagcccag tgtaaagtgg cccacggcgg acagagcgta
     6001 cagtgcgttc tccagggaga agccttgctg acacaggaac gcgagctgat tttccagggt
     6061 ttcgtactgt ttctctgttg ggcgggtgcc gagatgcact ttagccccgt cgcgatgtga
     6121 gaggagagca cagcggtatg acttggcgtt gttccgcaga aagtcttgcc atgactcgcc
     6181 ttccaggggg caggagtggg tatgatgcct gtccagcatc tcgattggca gggcatcgag
     6241 cagggcccgc ttgttcttca cgtgccagta cagggtaggc tgctcaactc ccagcttttg
     6301 agcgagtttc cttgtcgtca ggccttcgat accgactcca ttgagtaatt ccagagcaga
     6361 gtttatgatt ttgctcttgt ccagtctaga catatgaatt cggggccgcg gaggctggat
     6421 cggtcccggt gtcttctatg gaggtcaaaa cagcgtggat ggcgtctcca ggcgatctga
     6481 cggttcacta aacgagctct gcttatatag gcctcccacc gtacacgcct acctcgaccc
     6541 gggtaccgag ctcgactttc acttt
//
