LOCUS       sequence-376647-        5790 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector, high-level expression of EGFP in neuronal cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5790)
  AUTHORS   Sohn J, Takahashi M, Okamoto S, Ishida Y, Furuta T, Hioki H
  TITLE     A Single Vector Platform for High-Level Gene Transduction of Central
            Neurons: Adeno-Associated Virus Vector Equipped with the Tet-Off 
            System.
  JOURNAL   PLoS One. 2017 Jan 6;12(1):e0169611. doi: 
            10.1371/journal.pone.0169611. eCollection 2017.
  PUBMED    28060929
REFERENCE   2  (bases 1 to 5790)
  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: "PLoS One.";
            date: "2017-01-6"; volume: "12(1):e0169611. doi"; pages: " 
            10.1371/journal.pone.0169611. eCollection 2017"
COMMENT     Sequence Label: pAAV2-SynTetOff-GFP
FEATURES             Location/Qualifiers
     source          1..5790
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   9..138
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    153..177
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    277..398
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(314..333)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     368..387
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(422..430,431..466,467..469,470..505,
                     506..508,509..544,545..550,551..1168))
                     /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"
     promoter        1689..2003
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1697..1715
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1733..1751
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1768..1786
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1804..1822
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1840..1858
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1875..1893
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1911..1929
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1976..2000
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2035..2044
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2041..2043,2044..2046,2047..2760)
                     /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(2041..2043,2044..2046,2047..2757)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2086..2107)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2347..2366)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2694..2715
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2767..2991
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(3014..3038)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     repeat_region   3061..3201
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3061..3190
                     /label=AAV2 ITR
     rep_origin      3276..3731
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3363..3382)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3573..3594
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3748..3767)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3867..3889
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3927..3945)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4013..4117
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4118..4186,4187..4978)
                     /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(4336..4355)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5149..5737
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5638..5657
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcacatgtcc tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg
       61 cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact
      121 ccatcactag gggttcctgc ggccgcacgc gtacaagttt gtacaaaaaa gcaggctttg
      181 gatcttacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
      241 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
      301 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
      361 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgaga tccttactta
      421 gttacccggg gagcatgtca aggtcaaaat cgtcaagagc gtcagcaggc agcatatcaa
      481 ggtcaaagtc gtcaagggca tcggctggga gcatgtctaa gtcaaaatcg tcaagggcgt
      541 cggccggccc gccgctttcg cactttagct gtttctccag gccacatatg attagttcca
      601 ggccgaaaag gaaggcaggt tcggctccct gatggtcgaa cagctcaatt gcttgtctca
      661 gaagtggggg catagaatcg gtggtaggtg tctctctttc ctcttttgct acttgatgct
      721 cctgatcctc caatacgcag cccagtgtaa agtggcccac ggcggacaga gcgtacagtg
      781 cgttctccag ggagaagcct tgctgacaca ggaacgcgag ctgattttcc agggtttcgt
      841 actgtttctc tgttgggcgg gtgccgagat gcactttagc cccgtcgcga tgtgagagga
      901 gagcacagcg gaatgacttg gcgttgttcc gcagaaagtc ttgccatgac tcgccttcca
      961 gggggcagaa gtgggtatga tgcctgtcca gcatctcgat tgccagggca tcgagcaggg
     1021 cccgcttgtt cttcacgtgc cagtacaggg taggctgctc aactcccagc ttttgagcga
     1081 gtttccttgt cgtcaggcct tcgataccga cttcattgag taattccaga gcagagttta
     1141 tgactttgct cttgtccagt ctagacatgg tgaattcgat atcaagctta tcgataccgt
     1201 ccgccgcagc gcagatggtc gcgcccgtgc ccccctatct cgcgcctcgc gtggtgcggt
     1261 ccggctgggc cggcggcggc gcggacgcga ccaaggtggc cgggaagggg agtttgcggg
     1321 ggaccggcga gtgacgtcag cgcgccttca gtgctgaggc ggcggtggcg cgcgccgcca
     1381 ggcgggggcg aaggcactgt ccgcggtgct gaagctggca gtgcgcacgc gcctcgccgc
     1441 atcctgtttc ccctccccct ctctgatagg ggatgcgcaa tttggggaat gggggttggg
     1501 tgcttgtcca gtgggtcggg gtcggtcgtc aggtaggcac ccccaccccg cctcatcctg
     1561 gtcctaaaac ccacttgcac tcatacgcag ggccctctgc agctcgaccg cgccgccccg
     1621 ggcggctcgc tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg
     1681 gggggctcga gtttactccc tatcagtgat agagaacgta tgtcgagttt actccctatc
     1741 agtgatagag aacgatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgagtt
     1801 tactccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt gatagagaac
     1861 gtatgtcgag tttatcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag
     1921 tgatagagaa cgtatgtcga ggtaggcgtg tacggtggga ggcctatata agcagagctc
     1981 gtttagtgaa ccgtcagatc gcctggagaa ttcgagctcg gtacccgggg atccgccacc
     2041 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2101 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2161 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2221 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     2281 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2341 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2401 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2461 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     2521 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2581 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2641 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2701 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     2761 acgcgtctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     2821 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     2881 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2941 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg ggcggccgcg
     3001 tcgagatatc tagacccagc tttcttgtac aaagtggtca cgtgcggacc gagcggccgc
     3061 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     3121 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     3181 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     3241 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     3301 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     3361 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3421 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3481 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3541 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3601 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     3661 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     3721 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3781 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3841 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3901 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     3961 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     4021 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4081 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4141 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4201 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4261 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4321 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4381 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4441 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4501 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4561 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4621 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     4681 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     4741 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     4801 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     4861 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     4921 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     4981 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5041 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5101 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5161 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5221 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5281 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5341 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5401 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5461 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5521 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5581 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5641 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5701 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5761 acggttcctg gccttttgct ggccttttgc
//
