LOCUS       sequence-176825-        4787 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet/Dox-inducible expression of EGFP-SpvB (Salmonella SpvB 375-591) 
            in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4787)
  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 4787)
  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-SpvB
FEATURES             Location/Qualifiers
     source          1..4787
                     /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          1939..2004
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             2134..2154
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2579..2700
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2616..2635)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2670..2689
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2786..2803)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2957..3545)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3037..3056)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3716..4507,4508..4576))
                     /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     4339..4358
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4577..4681)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4749..4767
                     /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 gaggtaattc atctcgacca aaatcaaaat gggcgattgt agaggcatca
     1201 aagcagattc aagctctgag gtactattca gctcaagggt acagtgtgat taataaatat
     1261 ttacgtgggg atgattatcc tgaaacacag gcaaaagaaa ctctgctctc cagagactat
     1321 ctttccacaa atgaacccag tgatgaggag tttaaaaatg ccatgtcagt ttatataaat
     1381 gatattgcgg agggattaag ttcacttccc gaaacagatc acagagtcgt ataccggggc
     1441 ctgaagcttg ataagcccgc attatcggat gtgctgaagg aatacactac tataggtaat
     1501 ataataatag ataaagcttt tatgagtaca tcgccagata aggcatggat aaatgacact
     1561 attctcaaca tatacctaga aaaaggacat aaaggtagaa tactcggaga tgttgcacat
     1621 tttaagggag aggcagagat gcttttccct ccaaatacta aactcaaaat cgaaagcatt
     1681 gtaaattgtg gatcccaaga ctttgcaagc cagcttagta agctgagatt aagtgatgat
     1741 gcaactgctg acacaaacag gataaaaaga ataataaaca tgagggtact caactcatga
     1801 taagatccaa tgtaactgta ttcagcgatg acgaaattct tagctattgt aatactctag
     1861 aggatctttg tgaaggaacc ttacttctgt ggtgtgacat aattggacaa actacctaca
     1921 gagatttaaa gctctaaggt aaatataaaa tttttaagtg tataatgtgt taaactactg
     1981 attctaattg tttgtgtatt ttagattcca acctatggaa ctgatgaatg ggagcagtgg
     2041 tggaatgcct ttaatgagga aaacctgttt tgctcagaag aaatgccatc tagtgatgat
     2101 gaggctactg ctgactctca acattctact cctccaaaaa agaagagaaa ggtagaagac
     2161 cccaaggact ttccttcaga attgctaagt tttttgagtc atgctgtgtt tagtaataga
     2221 actcttgctt gctttgctat ttacaccaca aaggaaaaag ctgcactgct atacaagaaa
     2281 attatggaaa aatattctgt aacctttata agtaggcata acagttataa tcataacata
     2341 ctgttttttc ttactccaca caggcataga gtgtctgcta ttaataacta tgctcaaaaa
     2401 ttgtgtacct ttagcttttt aatttgtaaa ggggttaata aggaatattt gatgtatagt
     2461 gccttgacta gagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     2521 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     2581 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     2641 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     2701 tcatgtctgc ggctctagag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2761 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2821 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2881 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2941 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3001 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3061 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3121 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     3181 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3241 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     3301 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     3361 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     3421 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     3481 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     3541 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     3601 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     3661 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     3721 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3781 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3841 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3901 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3961 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     4021 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     4081 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     4141 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     4201 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     4261 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     4321 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     4381 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     4441 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     4501 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     4561 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     4621 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     4681 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     4741 cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattc
//
