LOCUS       sequence-176810-        4100 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet/Dox-inducible expression of EGFP in mammalian cells (control).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4100)
  AUTHORS   Harterink M, da Silva ME, Will L, Turan J, Ibrahim A, Lang AE, van 
            Battum EY, Pasterkamp RJ, Kapitein LC, Kudryashov D, Barres BA, 
            Hoogenraad CC, Zuchero JB
  TITLE     DeActs: genetically encoded tools for perturbing the actin 
            cytoskeleton in single cells.
  JOURNAL   Nat Methods. 2017 Apr 10. doi: 10.1038/nmeth.4257.
  PUBMED    28394337
REFERENCE   2  (bases 1 to 4100)
  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: "Nat 
            Methods. 2017 Apr 10. doi: 10.1038/nmeth.4257."
COMMENT     Sequence Label: pTetON-EGFP
FEATURES             Location/Qualifiers
     source          1..4100
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        4..382
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    12..30
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    48..66
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    84..102
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    120..138
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    156..174
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    192..210
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    228..246
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     291..315
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      388..397
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(394..396,397..399,400..1113)
                     /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(394..396,397..399,400..1110)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(439..460)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(700..719)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1047..1068
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          1252..1317
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1447..1467
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1892..2013
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1929..1948)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1983..2002
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2099..2116)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2270..2858)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2350..2369)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3029..3820,3821..3889))
                     /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     3652..3671
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3890..3994)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4062..4080
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagttta ctccctatca gtgatagaga acgtatgaag agtttactcc ctatcagtga
       61 tagagaacgt atgcagactt tactccctat cagtgataga gaacgtataa ggagtttact
      121 ccctatcagt gatagagaac gtatgaccag tttactccct atcagtgata gagaacgtat
      181 ctacagttta ctccctatca gtgatagaga acgtatatcc agtttactcc ctatcagtga
      241 tagagaacgt ataagcttta ggcgtgtacg gtgggcgcct ataaaagcag agctcgttta
      301 gtgaaccgtc agatcgcctg gagcaattcc acaacacttt tgtcttatac caactttccg
      361 taccacttcc taccctcgta aagtcgagcc accatggtga gcaagggcga ggagctgttc
      421 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
      481 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
      541 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
      601 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
      661 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
      721 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
      781 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
      841 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
      901 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
      961 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     1021 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1081 atcactctcg gcatggacga gctgtacaag tgataagatc caatgtaact gtattcagcg
     1141 atgacgaaat tcttagctat tgtaatactc tagaggatct ttgtgaagga accttacttc
     1201 tgtggtgtga cataattgga caaactacct acagagattt aaagctctaa ggtaaatata
     1261 aaatttttaa gtgtataatg tgttaaacta ctgattctaa ttgtttgtgt attttagatt
     1321 ccaacctatg gaactgatga atgggagcag tggtggaatg cctttaatga ggaaaacctg
     1381 ttttgctcag aagaaatgcc atctagtgat gatgaggcta ctgctgactc tcaacattct
     1441 actcctccaa aaaagaagag aaaggtagaa gaccccaagg actttccttc agaattgcta
     1501 agttttttga gtcatgctgt gtttagtaat agaactcttg cttgctttgc tatttacacc
     1561 acaaaggaaa aagctgcact gctatacaag aaaattatgg aaaaatattc tgtaaccttt
     1621 ataagtaggc ataacagtta taatcataac atactgtttt ttcttactcc acacaggcat
     1681 agagtgtctg ctattaataa ctatgctcaa aaattgtgta cctttagctt tttaatttgt
     1741 aaaggggtta ataaggaata tttgatgtat agtgccttga ctagagatca taatcagcca
     1801 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     1861 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     1921 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     1981 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgcggctcta gagctgcatt
     2041 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2101 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2161 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2221 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2281 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2341 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2401 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2461 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2521 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2581 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     2641 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     2701 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     2761 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     2821 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     2881 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     2941 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3001 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3061 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3121 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3181 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3241 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3301 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3361 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3421 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3481 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3541 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     3601 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     3661 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     3721 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     3781 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     3841 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     3901 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     3961 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4021 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     4081 cctttcgtct tcaagaattc
//
