LOCUS       sequence-376644-        5065 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry 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 5065)
  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 5065)
  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: pENTR1A-SynTetOff-GFP
FEATURES             Location/Qualifiers
     source          1..5065
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      116..202
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      294..321
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     329..352
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    371..470
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    570..691
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(607..626)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     661..680
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(715..723,724..759,760..762,763..798,
                     799..801,802..837,838..843,844..1461))
                     /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        1982..2296
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1990..2008
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2026..2044
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2061..2079
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2097..2115
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2133..2151
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2168..2186
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2204..2222
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2269..2293
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2328..2337
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2334..2336,2337..2339,2340..3053)
                     /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(2334..2336,2337..2339,2340..3050)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2379..2400)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2640..2659)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2987..3008
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3060..3284
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(3307..3406)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3518..3537)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3529..4338
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVSLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3596..3615)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4428..5016
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4917..4936
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcta
       61 gcatggatct cggggacgtc taactactaa gcgagagtag ggaactgcca ggcatcaaat
      121 aaaacgaaag gctcagtcgg aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
      181 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgtga agcaacggcc
      241 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaacta agcagaaggc
      301 catcctgacg gatggccttt ttgcgtttct acaaactctt cctgttagtt agttacttaa
      361 gctcgggccc caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat
      421 tgataagcaa tgctttttta taatgccaac tttgtacaaa aaagcaggct ttggatctta
      481 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
      541 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
      601 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
      661 gtggtttgtc caaactcatc aatgtatctt atcatgtctg agatccttac ttagttaccc
      721 ggggagcatg tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat caaggtcaaa
      781 gtcgtcaagg gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg cgtcggccgg
      841 cccgccgctt tcgcacttta gctgtttctc caggccacat atgattagtt ccaggccgaa
      901 aaggaaggca ggttcggctc cctgatggtc gaacagctca attgcttgtc tcagaagtgg
      961 gggcatagaa tcggtggtag gtgtctctct ttcctctttt gctacttgat gctcctgatc
     1021 ctccaatacg cagcccagtg taaagtggcc cacggcggac agagcgtaca gtgcgttctc
     1081 cagggagaag ccttgctgac acaggaacgc gagctgattt tccagggttt cgtactgttt
     1141 ctctgttggg cgggtgccga gatgcacttt agccccgtcg cgatgtgaga ggagagcaca
     1201 gcggaatgac ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt ccagggggca
     1261 gaagtgggta tgatgcctgt ccagcatctc gattgccagg gcatcgagca gggcccgctt
     1321 gttcttcacg tgccagtaca gggtaggctg ctcaactccc agcttttgag cgagtttcct
     1381 tgtcgtcagg ccttcgatac cgacttcatt gagtaattcc agagcagagt ttatgacttt
     1441 gctcttgtcc agtctagaca tggtgaattc gatatcaagc ttatcgatac cgtccgccgc
     1501 agcgcagatg gtcgcgcccg tgccccccta tctcgcgcct cgcgtggtgc ggtccggctg
     1561 ggccggcggc ggcgcggacg cgaccaaggt ggccgggaag gggagtttgc gggggaccgg
     1621 cgagtgacgt cagcgcgcct tcagtgctga ggcggcggtg gcgcgcgccg ccaggcgggg
     1681 gcgaaggcac tgtccgcggt gctgaagctg gcagtgcgca cgcgcctcgc cgcatcctgt
     1741 ttcccctccc cctctctgat aggggatgcg caatttgggg aatgggggtt gggtgcttgt
     1801 ccagtgggtc ggggtcggtc gtcaggtagg cacccccacc ccgcctcatc ctggtcctaa
     1861 aacccacttg cactcatacg cagggccctc tgcagctcga ccgcgccgcc ccgggcggct
     1921 cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt tgggggggct
     1981 cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata
     2041 gagaacgatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc
     2101 tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc
     2161 gagtttatcc ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga
     2221 gaacgtatgt cgaggtaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt
     2281 gaaccgtcag atcgcctgga gaattcgagc tcggtacccg gggatccgcc accatggtga
     2341 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     2401 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     2461 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     2521 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     2581 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     2641 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     2701 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     2761 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     2821 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     2881 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     2941 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     3001 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taaacgcgtc
     3061 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     3121 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3181 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3241 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atgggcggcc gcgtcgagat
     3301 atctagaccc agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt
     3361 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgcag
     3421 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     3481 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     3541 caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     3601 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag
     3661 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     3721 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     3781 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca
     3841 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     3901 tccctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     3961 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     4021 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     4081 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     4141 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     4201 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     4261 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     4321 ctcgatgagt ttttctaatc agaattggtt aattggttgt aacattattc agattgggcc
     4381 ccgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4441 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4501 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4561 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4621 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4681 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4741 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4801 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4861 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4921 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4981 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5041 cggttcctgg ccttttgctg gcctt
//
