LOCUS       sequence-360828-        6529 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV designed to express rtTA and eGFP from a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6529)
  TITLE     Ploski Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6529)
  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            "Unpublished"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: pAAV-CMV-rtTA-IRES-GFP-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..6529
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(68..656)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(148..167)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(827..1618,1619..1687))
                     /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     1450..1469
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1688..1792)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1860..1878
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1916..1938)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2038..2057
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2074..2529)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2211..2232)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2423..2442
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2604..2744
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2615..2744
                     /label=AAV2 ITR
     enhancer        2844..3223
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3224..3427
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3378..3398
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3424..3448
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(3530..4147,4148..4153,4154..4189,4190..4192,4193..4228,
                     4229..4231,4232..4267,4268..4276)
                     /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
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    4318..4890
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4485..4502)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4712..4731
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(4893..4895,4896..4898,4899..5612)
                     /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             join(4893..4895,4896..4898,4899..5609)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4938..4959)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5199..5218)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5546..5567
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5680..6268
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5733..5753)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6151..6162)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             6276..6293
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6349..6368)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   join(6406..6529,1..6)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 tgcaggacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
       61 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
      121 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
      181 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
      241 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
      301 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
      361 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
      421 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
      481 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
      541 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
      601 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
      661 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
      721 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
      781 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
      841 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
      901 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
      961 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     1021 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     1081 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     1141 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     1201 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     1261 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     1321 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     1381 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     1441 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     1501 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     1561 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     1621 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     1681 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     1741 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     1801 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     1861 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     1921 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     1981 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     2041 catcagagca gattgtactg agagtgcacc ataaaattgt aaacgttaat attttgttaa
     2101 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaatagacc gaaatcggca
     2161 aaatccctta taaatcaaaa gaatagcccg agatagagtt gagtgttgtt ccagtttgga
     2221 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     2281 agggcgatgg cccactacgt gaaccatcac ccaaatcaag ttttttgggg tcgaggtgcc
     2341 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     2401 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct aaggcgctgg
     2461 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     2521 agggcgcgta ctatggttgc tttgacgtat gcggtgtgaa ataccgcaca gatgcgtaag
     2581 gagaaaatac cgcatcaggc gcccctgcag gcagctgcgc gctcgctcgc tcactgaggc
     2641 cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg
     2701 agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgcggcc gctcggtcga
     2761 ggagcttggc ccattgcata cgttgtatcc atatcataat atgtacattt atattggctc
     2821 atgtccaaca ttaccgccat gttgacattg attattgact agttattaat agtaatcaat
     2881 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     2941 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     3001 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     3061 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     3121 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     3181 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     3241 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     3301 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     3361 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     3421 cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct
     3481 ccatagaaga caccgggacc gatccagcct ccgcggcccc gaattcacca tgtctagact
     3541 ggacaagagc aaagtcataa acggcgctct ggaattactc aatggagtcg gtatcgaagg
     3601 cctgacgaca aggaaactcg ctcaaaagct gggagttgag cagcctaccc tgtactggca
     3661 cgtgaagaac aagcgggccc tgctcgatgc cctgccaatc gagatgctgg acaggcatca
     3721 tacccacttc tgccccctgg aaggcgagtc atggcaagac tttctgcgga acaacgccaa
     3781 gtcattccgc tgtgctctcc tctcacatcg cgacggggct aaagtgcatc tcggcacccg
     3841 cccaacagag aaacagtacg aaaccctgga aaatcagctc gcgttcctgt gtcagcaagg
     3901 cttctccctg gagaacgcac tgtacgctct gtccgccgtg ggccacttta cactgggctg
     3961 cgtattggag gaacaggagc atcaagtagc aaaagaggaa agagagacac ctaccaccga
     4021 ttctatgccc ccacttctga gacaagcaat tgagctgttc gaccggcagg gagccgaacc
     4081 tgccttcctt ttcggcctgg aactaatcat atgtggcctg gagaaacagc taaagtgcga
     4141 aagcggcggg ccggccgacg cccttgacga ttttgactta gacatgctcc cagccgatgc
     4201 ccttgacgac tttgaccttg atatgctgcc tgctgacgct cttgacgatt ttgaccttga
     4261 catgctcccc gggtaactaa gtaaggatcc cccgggctgc aggaattcga tggccgcccc
     4321 ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc
     4381 gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa
     4441 acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat
     4501 gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac
     4561 aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg
     4621 cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt
     4681 tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg
     4741 gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac
     4801 atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga
     4861 cgtggtttcc ctttgaaaaa cacgataata ccatggtgag caagggcgag gagctgttca
     4921 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     4981 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     5041 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     5101 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     5161 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     5221 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     5281 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     5341 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     5401 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     5461 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     5521 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     5581 tcactctcgg catggacgag ctgtacaagt aatccggact cagatccagc gctgataacc
     5641 cagcctccac cacaaacaag gatgccggta ccggccgata atcaacctct ggattacaaa
     5701 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     5761 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     5821 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     5881 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     5941 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     6001 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     6061 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     6121 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     6181 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     6241 cggatctccc tttgggccgc ctccccgcat cggcccacca ccaccaccac cacatgtgaa
     6301 tgatccaagc ttatcggttt attgcagctt ataatggtta caaataaagc aatagcatca
     6361 caaatttcac aaataaagca tttttttcac tgcgcgtgcg gccgcaggaa cccctagtga
     6421 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     6481 tcgcccgacg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcc
//
