LOCUS       sequence-376649-        5868 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector, high-level expression of EGFP with a palmitoylation 
            signal derived from GAP-43 N-terminus (palGFP) in neuronal cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5868)
  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 5868)
  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-palGFP
FEATURES             Location/Qualifiers
     source          1..5868
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   12..141
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    156..180
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    280..401
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(317..336)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     371..390
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(425..433,434..469,470..472,473..508,
                     509..511,512..547,548..553,554..1171))
                     /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        1692..2006
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1700..1718
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1736..1754
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1771..1789
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1807..1825
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1843..1861
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1878..1896
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1914..1932
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1979..2003
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2116..2125
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2122..2124,2125..2127,2128..2841)
                     /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(2122..2124,2125..2127,2128..2838)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2167..2188)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2428..2447)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2775..2796
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2848..3072
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(3095..3119)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     repeat_region   3142..3282
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3142..3271
                     /label=AAV2 ITR
     rep_origin      3357..3812
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3444..3463)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3654..3675
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3829..3848)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3948..3970
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4008..4026)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4094..4198
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4199..4267,4268..5059)
                     /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(4417..4436)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5230..5818
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5719..5738
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgctcacatg tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtacaag tttgtacaaa aaagcaggct
      181 ttggatctta ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
      241 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
      301 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
      361 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg agatccttac
      421 ttagttaccc ggggagcatg tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat
      481 caaggtcaaa gtcgtcaagg gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg
      541 cgtcggccgg cccgccgctt tcgcacttta gctgtttctc caggccacat atgattagtt
      601 ccaggccgaa aaggaaggca ggttcggctc cctgatggtc gaacagctca attgcttgtc
      661 tcagaagtgg gggcatagaa tcggtggtag gtgtctctct ttcctctttt gctacttgat
      721 gctcctgatc ctccaatacg cagcccagtg taaagtggcc cacggcggac agagcgtaca
      781 gtgcgttctc cagggagaag ccttgctgac acaggaacgc gagctgattt tccagggttt
      841 cgtactgttt ctctgttggg cgggtgccga gatgcacttt agccccgtcg cgatgtgaga
      901 ggagagcaca gcggaatgac ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt
      961 ccagggggca gaagtgggta tgatgcctgt ccagcatctc gattgccagg gcatcgagca
     1021 gggcccgctt gttcttcacg tgccagtaca gggtaggctg ctcaactccc agcttttgag
     1081 cgagtttcct tgtcgtcagg ccttcgatac cgacttcatt gagtaattcc agagcagagt
     1141 ttatgacttt gctcttgtcc agtctagaca tggtgaattc gatatcaagc ttatcgatac
     1201 cgtccgccgc agcgcagatg gtcgcgcccg tgccccccta tctcgcgcct cgcgtggtgc
     1261 ggtccggctg ggccggcggc ggcgcggacg cgaccaaggt ggccgggaag gggagtttgc
     1321 gggggaccgg cgagtgacgt cagcgcgcct tcagtgctga ggcggcggtg gcgcgcgccg
     1381 ccaggcgggg gcgaaggcac tgtccgcggt gctgaagctg gcagtgcgca cgcgcctcgc
     1441 cgcatcctgt ttcccctccc cctctctgat aggggatgcg caatttgggg aatgggggtt
     1501 gggtgcttgt ccagtgggtc ggggtcggtc gtcaggtagg cacccccacc ccgcctcatc
     1561 ctggtcctaa aacccacttg cactcatacg cagggccctc tgcagctcga ccgcgccgcc
     1621 ccgggcggct cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt
     1681 tgggggggct cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct
     1741 atcagtgata gagaacgatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga
     1801 gtttactccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag
     1861 aacgtatgtc gagtttatcc ctatcagtga tagagaacgt atgtcgagtt tactccctat
     1921 cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg ggaggcctat ataagcagag
     1981 ctcgtttagt gaaccgtcag atcgcctgga gaattcgagc tcggtacccg gggatccgcc
     2041 accatgctgt gctgtatgcg aagaaccaaa caggttgaaa agaatgatga ggaccaaaag
     2101 atccaccggc cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg
     2161 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     2221 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     2281 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     2341 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     2401 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     2461 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     2521 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     2581 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     2641 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     2701 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     2761 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     2821 atggacgagc tgtacaagta aacgcgtctg tgccttctag ttgccagcca tctgttgttt
     2881 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     2941 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     3001 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     3061 tgggctctat gggcggccgc gtcgagatat ctagacccag ctttcttgta caaagtggtc
     3121 acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     3181 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     3241 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     3301 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     3361 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3421 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3481 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3541 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     3601 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3661 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     3721 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3781 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     3841 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     3901 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3961 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     4021 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     4081 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4141 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4201 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4261 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4321 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4381 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     4441 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     4501 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     4561 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     4621 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     4681 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     4741 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     4801 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     4861 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     4921 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     4981 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5041 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5101 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5161 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5221 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5281 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5341 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5401 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5461 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5521 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5581 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5641 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5701 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5761 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5821 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttt
//
