LOCUS       sequence-245701-        7505 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian linearizer gene circuit in a Flp-In expression system with
            a hTetR::EGFP fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
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 Apr 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="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    814..832
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    840..858
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     intron          1041..1613
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     regulatory      1625..1634
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2297..2299,2300..2302,2303..3016)
                     /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(2297..2299,2300..2302,2303..3013)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2342..2363)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2603..2622)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2950..2971
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3084..3104)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(3457..3474)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3463..3687
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3820..3839)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3971..4018
                     /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             4026..5048
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4026..5048
                     /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    5178..5299
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5215..5234)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5269..5288
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5348..5364)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5348..5364)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5361..5383)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5372..5388
                     /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(5396..5402,5403..5420,5421..5426))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5441..5462
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5579..5596)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5750..6338)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5830..5849)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6509..7300,7301..7369))
                     /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     7132..7151
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7370..7474)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcatccctat cagtgataga gatcagatct
      841 ccctatcagt gatagagagc tgttttgacc tccatagaag acaccgggac cgatccagcc
      901 tccggactct agcgtttaaa cttaagcttg gtacccgggg atcctctagg gcctctgagc
      961 tattccagaa gtagtgaaga ggcttttttg gaggcctagg cttttgcaaa aagctccgga
     1021 tcgatcctga gaacttcagg gtgagtttgg ggacccttga ttgttctttc tttttcgcta
     1081 ttgtaaaatt catgttatat ggagggggca aagttttcag ggtgttgttt agaatgggaa
     1141 gatgtccctt gtatcaccat ggaccctcat gataattttg tttctttcac tttctactct
     1201 gttgacaacc attgtctcct cttattttct tttcattttc tgtaactttt tcgttaaact
     1261 ttagcttgca tttgtaacga atttttaaat tcacttttgt ttatttgtca gattgtaagt
     1321 actttctcta atcacttttt tttcaaggca atcagggtat attatattgt acttcagcac
     1381 agttttagag aacaattgtt ataattaaat gataaggtag aatatttctg catataaatt
     1441 ctggctggcg tggaaatatt cttattggta gaaacaacta catcctggtc atcatcctgc
     1501 ctttctcttt atggttacaa tgatatacac tgtttgagat gaggataaaa tactctgagt
     1561 ccaaaccggg cccctctgct aaccatgttc atgccttctt ctttttccta caggtcctgc
     1621 aggcgccacc atgggttcta gactggacaa gagcaaagtc ataaactctg ctctggaatt
     1681 actcaatgaa gtcggtatcg aaggcctgac gacaaggaaa ctcgctcaaa agctgggagt
     1741 tgagcagcct accctgtact ggcacgtgaa gaacaagcgg gccctgctcg atgccctggc
     1801 aatcgagatg ctggacaggc atcataccca cttctgcccc ctggaaggcg agtcatggca
     1861 agactttctg cggaacaacg ccaagtcatt ccgctgtgct ctcctctcac atcgcgacgg
     1921 ggctaaagtg catctcggca cccgcccaac agagaaacag tacgaaaccc tggaaaatca
     1981 gctcgcgttc ctgtgtcagc aaggcttctc cctggagaac gcactgtacg ctctgtccgc
     2041 cgtgggccac tttacactgg gctgcgtatt ggaggatcag gagcatcaag tagcaaaaga
     2101 ggaaagagag acacctacca ccgattctat gcccccactt ctgagacaag caattgagct
     2161 gttcgaccat cagggagccg aacctgcctt ccttttcggc ctggaactaa tcatatgtgg
     2221 cctggagaaa cagctaaagt gcgaaagcgg cgggccaaaa aagaagagaa agggtgacgg
     2281 tgctggttta attaacatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2341 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2401 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2461 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     2521 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2581 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2641 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2701 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     2761 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     2821 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2881 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     2941 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     3001 cgagctgtac aagtaaaccg gtgcggccgc aatcaacctc tggattacaa aatttgtgaa
     3061 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     3121 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3181 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3241 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     3301 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     3361 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcc
     3421 tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     3481 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     3541 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     3601 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3661 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     3721 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3781 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3841 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3901 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3961 cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcct
     4021 tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     4081 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     4141 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag
     4201 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca
     4261 ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt
     4321 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg
     4381 atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag
     4441 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt
     4501 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg
     4561 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg
     4621 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg
     4681 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg
     4741 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc
     4801 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg
     4861 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg
     4921 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg
     4981 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa
     5041 aggaatagca cgtactacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5101 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5161 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5221 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5281 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     5341 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5401 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5461 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5521 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5581 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5641 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5701 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5761 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5821 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5881 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5941 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6001 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6061 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6121 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6181 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6241 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6301 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6361 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6421 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6481 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6541 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6601 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6661 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6721 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6781 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6841 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6901 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6961 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7021 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7081 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7141 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7201 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7261 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7321 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7381 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7441 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7501 acgtc
//
