LOCUS       sequence-176808-        6154 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet/Dox-inducible expression of DHRFdd-SpvB (Salmonella SpvB 
            375-591) plus constitutive expression of mCherry in mammalian cells 
            (Dual-promoter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6154)
  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 6154)
  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-DHFRdd-SpvB; CMV-mCherry
FEATURES             Location/Qualifiers
     source          1..6154
                     /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"
     CDS             400..876
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             1549..1575
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     intron          1717..1782
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1912..1932
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2357..2478
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2564..2581)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2735..3323)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2815..2834)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3494..4285,4286..4354))
                     /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     4117..4136
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4355..4459)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    4491..4612
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4735..5445)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5013..5032)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5282..5300
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      5442..5451
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    5453..5533
                     /label=MCS
                     /note="multiple cloning site"
     promoter        complement(5556..5759)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5585..5605)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5760..6063
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6116..6134
                     /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 aagtcgagga tccgccacca tgatcagtct gattgcggcg
      421 ttagcggtag attacgttat cggcatggaa aacgccatgc cgtggaacct gcctgccgat
      481 ctcgcctggt ttaaacgcaa caccttaaat aaacccgtga ttatgggccg ccatacctgg
      541 gaatcaatcg gtcgtccgtt gccaggacgc aaaaatatta tcctcagcag tcaaccgagt
      601 acggacgatc gcgtaacgtg ggtgaagtcg gtggatgaag ccatcgcggc gtgtggtgac
      661 gtaccagaaa tcatggtgat tggcggcggt cgcgttattg aacagttctt gccaaaagcg
      721 caaaaactgt atctgacgca tatcgacgca gaagtggaag gcgacaccca tttcccggat
      781 tacgagccgg atgactggga atcggtattc agcgagttcc acgatgctga tgcgcagaac
      841 tctcacagct attgctttga gattctggag cggcgagaat tcggatccgg atccggatcc
      901 ggaggtaatt catctcgacc aaaatcaaaa tgggcgattg tagaggcatc aaagcagatt
      961 caagctctga ggtactattc agctcaaggg tacagtgtga ttaataaata tttacgtggg
     1021 gatgattatc ctgaaacaca ggcaaaagaa actctgctct ccagagacta tctttccaca
     1081 aatgaaccca gtgatgagga gtttaaaaat gccatgtcag tttatataaa tgatattgcg
     1141 gagggattaa gttcacttcc cgaaacagat cacagagtcg tataccgggg cctgaagctt
     1201 gataagcccg cattatcgga tgtgctgaag gaatacacta ctataggtaa tataataata
     1261 gataaagctt ttatgagtac atcgccagat aaggcatgga taaatgacac tattctcaac
     1321 atatacctag aaaaaggaca taaaggtaga atactcggag atgttgcaca ttttaaggga
     1381 gaggcagaga tgcttttccc tccaaatact aaactcaaaa tcgaaagcat tgtaaattgt
     1441 ggatcccaag actttgcaag ccagcttagt aagctgagat taagtgatga tgcaactgct
     1501 gacacaaaca ggataaaaag aataataaac atgagggtac tcaactcata cccatacgat
     1561 gttccagatt acgcttgata agatccaatg taactgtatt cagcgatgac gaaattctta
     1621 gctattgtaa tactctagag gatctttgtg aaggaacctt acttctgtgg tgtgacataa
     1681 ttggacaaac tacctacaga gatttaaagc tctaaggtaa atataaaatt tttaagtgta
     1741 taatgtgtta aactactgat tctaattgtt tgtgtatttt agattccaac ctatggaact
     1801 gatgaatggg agcagtggtg gaatgccttt aatgaggaaa acctgttttg ctcagaagaa
     1861 atgccatcta gtgatgatga ggctactgct gactctcaac attctactcc tccaaaaaag
     1921 aagagaaagg tagaagaccc caaggacttt ccttcagaat tgctaagttt tttgagtcat
     1981 gctgtgttta gtaatagaac tcttgcttgc tttgctattt acaccacaaa ggaaaaagct
     2041 gcactgctat acaagaaaat tatggaaaaa tattctgtaa cctttataag taggcataac
     2101 agttataatc ataacatact gttttttctt actccacaca ggcatagagt gtctgctatt
     2161 aataactatg ctcaaaaatt gtgtaccttt agctttttaa tttgtaaagg ggttaataag
     2221 gaatatttga tgtatagtgc cttgactaga gatcataatc agccatacca catttgtaga
     2281 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     2341 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     2401 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     2461 actcatcaat gtatcttatc atgtctgcgg ctctagagct gcattaatga atcggccaac
     2521 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2581 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2641 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2701 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2761 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2821 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2881 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2941 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3001 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3061 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3121 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     3181 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3241 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3301 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3361 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3421 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3481 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3541 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3601 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     3661 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3721 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3781 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     3841 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     3901 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     3961 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     4021 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     4081 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     4141 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     4201 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     4261 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     4321 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     4381 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4441 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgcgcct taagatacat
     4501 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     4561 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     4621 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
     4681 gtaaaacctc tacaaatgtg gtatggctga ttatgatcta gagtcgcggc cgctttactt
     4741 gtacagctcg tccatgccgc cggtggagtg gcggccctcg gcgcgttcgt actgttccac
     4801 gatggtgtag tcctcgttgt gggaggtgat gtccaacttg atgttgacgt tgtaggcgcc
     4861 gggcagctgc acgggcttct tggccttgta ggtggtcttg acctcagcgt cgtagtggcc
     4921 gccgtccttc agcttcagcc tctgcttgat ctcgcccttc agggcgccgt cctcggggta
     4981 catccgctcg gaggaggcct cccagcccat ggtcttcttc tgcattacgg ggccgtcgga
     5041 ggggaagttg gtgccgcgca gcttcacctt gtagatgaac tcgccgtcct gcagggagga
     5101 gtcctgggtc acggtcacca cgccgccgtc ctcgaagttc atcacgcgct cccacttgaa
     5161 gccctcgggg aaggacagct tcaagtagtc ggggatgtcg gcggggtgct tcacgtaggc
     5221 cttggagccg tacatgaact gaggggacag gatgtcccag gcgaagggca gggggccacc
     5281 cttggtcacc ttcagcttgg cggtctgggt gccctcgtag gggcggccct cgccctcgcc
     5341 ctcgatctcg aactcgtggc cgttcacgga gccctccatg tgcaccttga agcgcatgaa
     5401 ctccttgatg atggccatgt tatcctcctc gcccttgctc accatggtgg cgaccggtgg
     5461 atcccgggcc cgcggtaccg tcgactgcag aattcgaagc ttgagctcga gatctgagtc
     5521 cggtagcgct agcggatctg acggttcact aaaccagctc tgcttatata gacctcccac
     5581 cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag
     5641 tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg gagacttgga
     5701 aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa ccgcatcacc
     5761 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc
     5821 atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta
     5881 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
     5941 cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt cattattgac
     6001 gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt aagttatgta
     6061 acgcggaact ccatatatct aagaaaccat tattatcatg acattaacct ataaaaatag
     6121 gcgtatcacg aggccctttc gtcttcaaga attc
//
