LOCUS       sequence-176821-        4511 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet/Dox-inducible expression of EGFP-GS1 (Human Gelsolin 53-176) in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4511)
  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 4511)
  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-DeAct-GS1
FEATURES             Location/Qualifiers
     source          1..4511
                     /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..1110)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(394..396,397..399,400..1110)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /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          1663..1728
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1858..1878
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2303..2424
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2340..2359)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2394..2413
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2510..2527)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2681..3269)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2761..2780)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3440..4231,4232..4300))
                     /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     4063..4082
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4301..4405)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4473..4491
                     /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 tccggactca gatctcgagc tcaagcttcg
     1141 aattctatgg tggtggaaca ccccgagttc ctcaaggcag ggaaggagcc tggcctgcag
     1201 atctggcgtg tggagaagtt cgatctggtg cccgtgccca ccaaccttta tggagacttc
     1261 ttcacgggcg acgcctacgt catcctgaag acagtgcagc tgaggaacgg aaatctgcag
     1321 tatgacctcc actactggct gggcaatgag tgcagccagg atgagagcgg ggcggccgcc
     1381 atctttaccg tgcagctgga tgactacctg aacggccggg ccgtgcagca ccgtgaggtc
     1441 cagggcttcg agtcggccac cttcctaggc tacttcaagt ctggcctgaa gtacaagaaa
     1501 ggaggtgtgg catcaggatt ctgataagat ccaatgtaac tgtattcagc gatgacgaaa
     1561 ttcttagcta ttgtaatact ctagaggatc tttgtgaagg aaccttactt ctgtggtgtg
     1621 acataattgg acaaactacc tacagagatt taaagctcta aggtaaatat aaaattttta
     1681 agtgtataat gtgttaaact actgattcta attgtttgtg tattttagat tccaacctat
     1741 ggaactgatg aatgggagca gtggtggaat gcctttaatg aggaaaacct gttttgctca
     1801 gaagaaatgc catctagtga tgatgaggct actgctgact ctcaacattc tactcctcca
     1861 aaaaagaaga gaaaggtaga agaccccaag gactttcctt cagaattgct aagttttttg
     1921 agtcatgctg tgtttagtaa tagaactctt gcttgctttg ctatttacac cacaaaggaa
     1981 aaagctgcac tgctatacaa gaaaattatg gaaaaatatt ctgtaacctt tataagtagg
     2041 cataacagtt ataatcataa catactgttt tttcttactc cacacaggca tagagtgtct
     2101 gctattaata actatgctca aaaattgtgt acctttagct ttttaatttg taaaggggtt
     2161 aataaggaat atttgatgta tagtgccttg actagagatc ataatcagcc ataccacatt
     2221 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     2281 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     2341 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     2401 gtccaaactc atcaatgtat cttatcatgt ctgcggctct agagctgcat taatgaatcg
     2461 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     2521 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2581 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2641 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2701 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2761 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2821 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2881 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2941 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3001 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3061 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3121 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3181 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3241 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3301 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3361 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3421 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3481 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3541 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3601 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3661 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3721 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     3781 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3841 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3901 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     3961 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4021 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4081 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4141 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4201 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4261 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4321 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4381 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     4441 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     4501 ttcaagaatt c
//
