LOCUS       sequence-381567-        5853 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector, high-level expression of EGFP with a 
            myristoylation/palmitoylation signal derived from Fyn N-terminus 
            (myrGFP) in neuronal cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5853)
  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 5853)
  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-myrGFP
FEATURES             Location/Qualifiers
     source          1..5853
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   2..131
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    146..170
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    270..391
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(307..326)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     361..380
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(415..423,424..459,460..462,463..498,
                     499..501,502..537,538..543,544..1161))
                     /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        1682..1996
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1690..1708
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1726..1744
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1761..1779
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1797..1815
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1833..1851
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1868..1886
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1904..1922
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1969..1993
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2028..2037
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2091..2100
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2097..2099,2100..2102,2103..2816)
                     /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(2097..2099,2100..2102,2103..2813)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2142..2163)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2403..2422)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2750..2771
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2823..3047
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(3070..3094)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     repeat_region   3117..3257
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3117..3246
                     /label=AAV2 ITR
     rep_origin      3332..3787
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3419..3438)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3629..3650
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3804..3823)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3923..3945
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3983..4001)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4069..4173
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4174..4242,4243..5034)
                     /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(4392..4411)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5205..5793
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5694..5713
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc gggcgacctt
       61 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      121 taggggttcc tgcggccgca cgcgtacaag tttgtacaaa aaagcaggct ttggatctta
      181 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
      241 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
      301 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
      361 gtggtttgtc caaactcatc aatgtatctt atcatgtctg agatccttac ttagttaccc
      421 ggggagcatg tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat caaggtcaaa
      481 gtcgtcaagg gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg cgtcggccgg
      541 cccgccgctt tcgcacttta gctgtttctc caggccacat atgattagtt ccaggccgaa
      601 aaggaaggca ggttcggctc cctgatggtc gaacagctca attgcttgtc tcagaagtgg
      661 gggcatagaa tcggtggtag gtgtctctct ttcctctttt gctacttgat gctcctgatc
      721 ctccaatacg cagcccagtg taaagtggcc cacggcggac agagcgtaca gtgcgttctc
      781 cagggagaag ccttgctgac acaggaacgc gagctgattt tccagggttt cgtactgttt
      841 ctctgttggg cgggtgccga gatgcacttt agccccgtcg cgatgtgaga ggagagcaca
      901 gcggaatgac ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt ccagggggca
      961 gaagtgggta tgatgcctgt ccagcatctc gattgccagg gcatcgagca gggcccgctt
     1021 gttcttcacg tgccagtaca gggtaggctg ctcaactccc agcttttgag cgagtttcct
     1081 tgtcgtcagg ccttcgatac cgacttcatt gagtaattcc agagcagagt ttatgacttt
     1141 gctcttgtcc agtctagaca tggtgaattc gatatcaagc ttatcgatac cgtccgccgc
     1201 agcgcagatg gtcgcgcccg tgccccccta tctcgcgcct cgcgtggtgc ggtccggctg
     1261 ggccggcggc ggcgcggacg cgaccaaggt ggccgggaag gggagtttgc gggggaccgg
     1321 cgagtgacgt cagcgcgcct tcagtgctga ggcggcggtg gcgcgcgccg ccaggcgggg
     1381 gcgaaggcac tgtccgcggt gctgaagctg gcagtgcgca cgcgcctcgc cgcatcctgt
     1441 ttcccctccc cctctctgat aggggatgcg caatttgggg aatgggggtt gggtgcttgt
     1501 ccagtgggtc ggggtcggtc gtcaggtagg cacccccacc ccgcctcatc ctggtcctaa
     1561 aacccacttg cactcatacg cagggccctc tgcagctcga ccgcgccgcc ccgggcggct
     1621 cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt tgggggggct
     1681 cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata
     1741 gagaacgatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc
     1801 tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc
     1861 gagtttatcc ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga
     1921 gaacgtatgt cgaggtaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt
     1981 gaaccgtcag atcgcctgga gaattcgagc tcggtacccg gggatctgcc accatgggct
     2041 gtgtgcaatg taaggataaa gaagcaacaa aactgacggg gggatccatc gccaccatgg
     2101 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     2161 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     2221 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2281 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     2341 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2401 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2461 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2521 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2581 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2641 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2701 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2761 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaaacgc
     2821 gtctgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2881 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2941 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3001 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatgggcg gccgcgtcga
     3061 gatatctaga cccagctttc ttgtacaaag tggtcacgtg cggaccgagc ggccgcagga
     3121 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     3181 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     3241 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3301 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     3361 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     3421 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3481 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3541 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3601 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3661 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     3721 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     3781 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     3841 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3901 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3961 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     4021 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4081 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4141 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     4201 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     4261 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     4321 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     4381 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     4441 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     4501 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     4561 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     4621 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     4681 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     4741 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     4801 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     4861 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     4921 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     4981 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     5041 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5101 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     5161 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5221 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     5281 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5341 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     5401 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5461 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5521 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5581 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5641 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5701 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5761 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5821 ttcctggcct tttgctggcc ttttgctcac atg
//
