LOCUS       sequence-248946-        6246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian positive feedback synthetic gene circuit within a Flp-In 
            expression vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6246)
  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 6246)
  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: pKF-P14MM2AG5h
FEATURES             Location/Qualifiers
     source          1..6246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(75..94)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        287..601
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    295..313
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    331..349
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    366..384
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    402..420
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    438..456
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    473..491
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    509..527
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     574..598
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(606..1223,1224..1229,1230..1265,1266..1268,1269..1304,
                     1305..1307,1308..1343,1344..1349)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             1359..1415
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(1416..1418,1419..1421,1422..2135)
                     /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(1416..1418,1419..1421,1422..2132)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1461..1482)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1722..1741)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2069..2090
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2229..2246)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2235..2459
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    2743..2790
                     /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             2798..3820
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2798..3820
                     /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    3950..4071
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3987..4006)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4041..4060
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4120..4136)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4120..4136)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4133..4155)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4144..4160
                     /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(4168..4174,4175..4192,4193..4198))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4213..4234
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4351..4368)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4522..5110)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4602..4621)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5281..6072,6073..6141))
                     /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     5904..5923
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6142..6246)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
       61 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
      121 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
      181 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
      241 gatgtacggg ccagatatac gcgtcgaggc cctttcgtct tcactcgagt ttactcccta
      301 tcagtgatag agaacgtatg tcgagtttac tccctatcag tgatagagaa cgatgtcgag
      361 tttactccct atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga
      421 acgtatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgagtt tatccctatc
      481 agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg tatgtcgagg
      541 taggcgtgta cggtgggagg cctatataag cagagctcgt ttagtgaacc gtcagatcgc
      601 ctggaatgtc tagactggac aagagcaaag tcataaacgg cgctctggaa ttactcaatg
      661 gagtcggtat cgaaggcctg acgacaagga aactcgctca aaagctggga gttgagcagc
      721 ctaccctgta ctggcacgtg aagaacaagc gggccctgct cgatgccctg ccaatcgaga
      781 tgctggacag gcatcatacc cacttctgcc ccctggaagg cgagtcatgg caagactttc
      841 tgcggaacaa cgccaagtca ttccgctgtg ctctcctctc acatcgcgac ggggctaaag
      901 tgcatctcgg cacccgccca acagagaaac agtacgaaac cctggaaaat cagctcgcgt
      961 tcctgtgtca gcaaggcttc tccctggaga acgcactgta cgctctgtcc gccgtgggcc
     1021 actttacact gggctgcgta ttggaggaac aggagcatca agtagcaaaa gaggaaagag
     1081 agacacctac caccgattct atgcccccac ttctgagaca agcaattgag ctgttcgacc
     1141 ggcagggagc cgaacctgcc ttccttttcg gcctggaact aatcatatgt ggcctggaga
     1201 aacagctaaa gtgcgaaagc ggcgggccgg ccgacgccct tgacgatttt gacttagaca
     1261 tgctcccagc cgatgccctt gacgactttg accttgatat gctgcctgct gacgctcttg
     1321 acgattttga ccttgacatg ctccccgggg gaagcggagc tactaacttc agcctgctga
     1381 agcaggctgg agacgtggag gagaaccctg gacctatggt gagcaagggc gaggagctgt
     1441 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1501 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     1561 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     1621 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1681 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     1741 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1801 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     1861 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     1921 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     1981 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     2041 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     2101 ggatcactct cggcatggac gagctgtaca agtaaggatc cactagtcca gtgtggtgga
     2161 attctgcaga tatccagcac agtggcggcc gctcgagtct agagggcccg tttaaacccg
     2221 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2281 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2341 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2401 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     2461 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     2521 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2581 cctagcgccc gctcctctcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2641 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2701 cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag
     2761 ttcctattct ctagaaagta taggaacttc cttggccaaa aagcctgaac tcaccgcgac
     2821 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc
     2881 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg
     2941 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc
     3001 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta
     3061 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc
     3121 cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca
     3181 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga
     3241 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac
     3301 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc
     3361 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg
     3421 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt
     3481 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt
     3541 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat
     3601 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc
     3661 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat
     3721 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg
     3781 aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtactac gagatttcga
     3841 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     3901 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     3961 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4021 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4081 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4141 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4201 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4261 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4321 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4381 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggctat ccacagaatc
     4441 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4501 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4561 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4621 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4681 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4741 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4801 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4861 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4921 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     4981 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5041 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5101 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5161 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5221 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5281 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5341 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5401 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5461 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5521 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5581 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5641 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5701 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5761 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5821 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5881 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     5941 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6001 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6061 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6121 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6181 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6241 tccgcg
//
