LOCUS       sequence-248947-        7505 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Mammalian linearizer gene circuit in a Flp-In expression system with
            a hTetR::EGFP fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7505)
  AUTHORS   Farquhar KS, Charlebois DA, Szenk M, Cohen J, Nevozhay D, Balazsi G
  TITLE     Role of network-mediated stochasticity in mammalian drug resistance.
  JOURNAL   Nat Commun. 2019 Jun 24;10(1):2766. doi: 10.1038/s41467-019-10330-w.
  PUBMED    31235692
REFERENCE   2  (bases 1 to 7505)
  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 
            Commun."; date: "2019-06-24"; volume: "10(1):2766. doi"; pages: " 
            10.1038/s41467-019-10330-w"
COMMENT     Sequence Label: pDN-D2irTNG5kwh
FEATURES             Location/Qualifiers
     source          1..7505
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    200..218
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    226..244
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     intron          427..999
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     regulatory      1011..1020
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1683..1685,1686..1688,1689..2402)
                     /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(1683..1685,1686..1688,1689..2399)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1728..1749)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1989..2008)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2336..2357
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2470..2490)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(2843..2860)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2849..3073
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3206..3225)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3357..3404
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3412..4434
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3412..4434
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4564..4685
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4601..4620)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4655..4674
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4734..4750)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4734..4750)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4747..4769)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4758..4774
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4782..4788,4789..4806,4807..4812))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4827..4848
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4965..4982)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5136..5724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5216..5235)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5895..6686,6687..6755))
                     /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     6518..6537
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6756..6860)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6935..6954)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7126..7505
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcat ccctatcagt gatagagatc agatctccct atcagtgata
      241 gagagctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctagcg
      301 tttaaactta agcttggtac ccggggatcc tctagggcct ctgagctatt ccagaagtag
      361 tgaagaggct tttttggagg cctaggcttt tgcaaaaagc tccggatcga tcctgagaac
      421 ttcagggtga gtttggggac ccttgattgt tctttctttt tcgctattgt aaaattcatg
      481 ttatatggag ggggcaaagt tttcagggtg ttgtttagaa tgggaagatg tcccttgtat
      541 caccatggac cctcatgata attttgtttc tttcactttc tactctgttg acaaccattg
      601 tctcctctta ttttcttttc attttctgta actttttcgt taaactttag cttgcatttg
      661 taacgaattt ttaaattcac ttttgtttat ttgtcagatt gtaagtactt tctctaatca
      721 cttttttttc aaggcaatca gggtatatta tattgtactt cagcacagtt ttagagaaca
      781 attgttataa ttaaatgata aggtagaata tttctgcata taaattctgg ctggcgtgga
      841 aatattctta ttggtagaaa caactacatc ctggtcatca tcctgccttt ctctttatgg
      901 ttacaatgat atacactgtt tgagatgagg ataaaatact ctgagtccaa accgggcccc
      961 tctgctaacc atgttcatgc cttcttcttt ttcctacagg tcctgcaggc gccaccatgg
     1021 gttctagact ggacaagagc aaagtcataa actctgctct ggaattactc aatgaagtcg
     1081 gtatcgaagg cctgacgaca aggaaactcg ctcaaaagct gggagttgag cagcctaccc
     1141 tgtactggca cgtgaagaac aagcgggccc tgctcgatgc cctggcaatc gagatgctgg
     1201 acaggcatca tacccacttc tgccccctgg aaggcgagtc atggcaagac tttctgcgga
     1261 acaacgccaa gtcattccgc tgtgctctcc tctcacatcg cgacggggct aaagtgcatc
     1321 tcggcacccg cccaacagag aaacagtacg aaaccctgga aaatcagctc gcgttcctgt
     1381 gtcagcaagg cttctccctg gagaacgcac tgtacgctct gtccgccgtg ggccacttta
     1441 cactgggctg cgtattggag gatcaggagc atcaagtagc aaaagaggaa agagagacac
     1501 ctaccaccga ttctatgccc ccacttctga gacaagcaat tgagctgttc gaccatcagg
     1561 gagccgaacc tgccttcctt ttcggcctgg aactaatcat atgtggcctg gagaaacagc
     1621 taaagtgcga aagcggcggg ccaaaaaaga agagaaaggg tgacggtgct ggtttaatta
     1681 acatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1741 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1801 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1861 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1921 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1981 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2041 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     2101 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     2161 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     2221 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2281 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     2341 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     2401 aaaccggtgc ggccgcaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     2461 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     2521 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     2581 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     2641 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     2701 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     2761 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcctcga gtctagaggg
     2821 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     2881 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     2941 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     3001 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     3061 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     3121 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3181 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3241 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3301 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tacctagaag
     3361 ttcctattcc gaagttccta ttctctagaa agtataggaa cttccttggc caaaaagcct
     3421 gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac
     3481 ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt
     3541 ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat
     3601 cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc
     3661 gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct
     3721 gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg
     3781 gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
     3841 actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact
     3901 gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg
     3961 gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc
     4021 ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat
     4081 tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag
     4141 cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg
     4201 tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat
     4261 gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc
     4321 gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta
     4381 ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgta
     4441 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4501 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4561 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4621 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4681 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     4741 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4801 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4861 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4921 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4981 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5041 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5101 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5161 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5221 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5281 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5341 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5401 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5461 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5521 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5581 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5641 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5701 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5761 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5821 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5881 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5941 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6001 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6061 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6121 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6181 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6241 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6301 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6361 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6421 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6481 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6541 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6601 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6661 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6721 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6781 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6841 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
     6901 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     6961 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     7021 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     7081 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     7141 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     7201 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     7261 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     7321 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     7381 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     7441 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     7501 ccatg
//
