LOCUS       sequence-347269-        8469 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TF C self-activation cassette in MultiFate-3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8469)
  AUTHORS   Zhu R, Del Rio-Salgado JM, Garcia-Ojalvo J, Elowitz MB
  TITLE     Synthetic multistability in mammalian cells.
  JOURNAL   Science. 2022 Jan 21;375(6578):eabg9765. doi: 
            10.1126/science.abg9765. Epub 2022 Jan 21.
  PUBMED    35050677
REFERENCE   2  (bases 1 to 8469)
  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: "Science."; 
            date: "2022-01-21"; volume: "375(6578):eabg9765. doi"; pages: " 
            10.1126/science.abg9765. Epub 2022 Jan 21"
COMMENT     Sequence Label: 
            MF85_PB-14xUAS-12x42bs-10x(ErbB2bs_ErbB2bs)-miniCMV-NLS-FKBP12F36V-E
            rbB2ZFR2AR39A-VP16-NLS-DHFR-IRES-mTurquoise2-PEST-BGHpA
FEATURES             Location/Qualifiers
     source          1..8469
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     2177..2201
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2285..2294
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2297..2317
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2321..2641
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=DmrB
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             3164..3184
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3194..3217
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    join(3713..4287,4288..4290,4291..4299)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3880..3897)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4107..4126
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(4300..4302,4303..4305,4306..5016)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4345..4366)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4606..4625)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4953..4974
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5177..5194)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5183..5390
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5923..5957
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6448..6464)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6448..6464)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6461..6483)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6472..6488
                     /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(6496..6502,6503..6520,6521..6526))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6541..6562
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6679..6696)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6850..7438)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6930..6949)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7609..8400,8401..8469))
                     /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     8232..8251
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc acggccacta gtggcctcca cggccacaag
     1381 cttcatatgc ggagtactgt cctccgggct ggcggagtac tgtcctccgg caaggtcgga
     1441 gtactgtcct ccgacactag aggtcggagt actgtcctcc gacgcaaggc ggagtactgt
     1501 cctccgggct gcggagtact gtcctccggc aaggtcggag tactgtcctc cgacactaga
     1561 ggtcggagta ctgtcctccg acgcaaggtc ggagtactgt cctccgacac tagaggtcgg
     1621 agtactgtcc tccgacgcaa ggtcggagta ctgtcctccg acactagagg tcggagtact
     1681 gtcctccgac gcaaggcgga gtactgtcct ccgggctggc ggagtactgt cctccggcaa
     1741 gggctcgacg ctgctgacgc tgctgacgct gctgacgctg ctgacgctgc tgacgctgct
     1801 gacgctgctg acgctgctga cgctgctgac gctgctgacg ctgctcaatt gtcgccggcg
     1861 gccgcagtgg ccgcagtgac gtatatgccg cagtggccgc agtgtctaga gcgccgcagt
     1921 ggccgcagtg atcgatatgc cgcagtggcc gcagtgtcta gagcgccgca gtggccgcag
     1981 tgtctagagc gccgcagtgg ccgcagtgat cgatatgccg cagtggccgc agtgtcacgt
     2041 acgccgcagt ggccgcagtg tatgacctgc cgcagtggcc gcagtgacgt atatgccgca
     2101 gtggccgcag tgcctagacc ggttgataga gaacgtataa gctttaggcg tgtacggtgg
     2161 gcgcctataa aagcagagct cgtttagtga accgtcagat cgcctggagc aattccacaa
     2221 cacttttgtc ttataccaac tttccgtacc acttcctacc ctcgtaaatc gacgaagctt
     2281 ctaggccacc atgggaccca agaaaaagcg gaaggtgtcc ggagtgcagg tggagactat
     2341 ctccccagga gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac
     2401 cgggatgctt gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa
     2461 gtttatgcta ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag
     2521 tgtgggtcag agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca
     2581 cccaggcatc atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga
     2641 aggatccgag gccccattcc agtgcagaat ctgcatgcgg aacttctcca gatctgacgt
     2701 gctggccaac cacaccagaa cacacacagg cgagaagccc ttccagtgcc ggatctgtat
     2761 ggccaacttt tcccagtcct ccacactgac ccggcacctg agaacccata ccggcgaaaa
     2821 gccttttcag tgtcgcatct gcatgagaaa ctttagcgag cggcagggcc tgaagagaca
     2881 tctgaaaacc cacactggcg agaaaggcgg cggatcctct ggcgctcctc ctaccgatgt
     2941 gtctctggga gatgagctgc acctggacgg cgaggatgtg gctatggctc atgccgacgc
     3001 tctggacgac ttcgacttgg atatgctcgg cgacggcgac tctcctggac ctggctttac
     3061 ccctcatgac tctgcccctt acggcgccct ggatatggcc gatttcgagt tcgagcagat
     3121 gttcaccgat gctctgggca tcgatgagta cggcggaggc tcccccaaga aaaagcggaa
     3181 ggtgggctcg agcgactaca aggacgacga cgacaagtcg agcatctccc tgatcgctgc
     3241 cctggctgtg gattacgtga tcggaatgga aaacgccatg ccatggaacc tgcctgctga
     3301 cctggcctgg ttcaagcgga acaccctgaa caagcccgtg atcatgggca gacacacctg
     3361 ggagtctatc ggcagacctc tgcctggccg gaagaacatc atcctgtcct ctcagccttc
     3421 caccgacgac agagtgacct gggtcaagtc tgtggacgag gctatcgctg cttgtggcga
     3481 cgtgccagag atcatggtta tcggcggagg cagagtgatc gagcagttcc tgcctaaggc
     3541 tcagaagctg tatctgaccc acatcgacgc cgaagtggaa ggcgataccc actttcctga
     3601 ctacgagccc gacgactggg agtccgtgtt ctctgagttc cacgacgccg atgctcagaa
     3661 ctcccactcc tactgcttcg agatcctgga gcggagatag gtcgaccaga tccccctctc
     3721 cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg
     3781 tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg
     3841 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag
     3901 gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt
     3961 ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc
     4021 aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga
     4081 gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga
     4141 aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct
     4201 ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg
     4261 ttttcctttg aaaaacacga tgataatatg gccacaacca tggtgagcaa gggcgaggag
     4321 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     4381 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     4441 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgtcctgg
     4501 ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     4561 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     4621 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     4681 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacttt
     4741 agcgacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag
     4801 atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca gcagaacacc
     4861 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccaag
     4921 ttgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     4981 gccgggatca ctctcggcat ggacgagctg tacaagtccg gcaagcttag ccatggcttc
     5041 ccgccggagg tggaggagca ggatgatggc acgctgccca tgtcttgtgc ccaggagagc
     5101 gggatggacc gtcaccctgc agcctgtgct tctgctagga tcaatgtgtg agctagcgaa
     5161 ttcgaattta aatcggcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     5221 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     5281 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     5341 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga cgcggccgcg
     5401 ggcccattgg tatggctttt tccccgtatc cccccaggtg tctgcaggct caaagagcag
     5461 cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg
     5521 cacgctgccg gctcggggat gcggggggag cgccggaccg gagcggagcc ccgggcggct
     5581 cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt tggggggggg
     5641 ctgtccctga tatctataac aagaaaatat atatataata agttatcacg taagtagaac
     5701 atgaaataac aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc
     5761 atgcgtcatt ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg
     5821 acaagcacgc ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg
     5881 gattcgcgct atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc
     5941 agactatctt tctagggtta atctagctgc atcaggatca tatcgtcggg tcttttttcc
     6001 ggctcagtca tcgcccaagc tggcgctatc tgggcatcgg ggaggaagaa gcccgtgcct
     6061 tttcccgcga ggttgaagcg gcatggaaag agtttgccga ggatgactgc tgctgcattg
     6121 acgttgagcg aaaacgcacg tttaccatga tgattcggga aggtgtggcc atgcacgcct
     6181 ttaacggtga actgttcgtt caggccacct gggataccag ttcgtcgcgg cttttccgga
     6241 cacagttccg gatggtcagc ccgaagcgca tcagcaaccc gaacaatacc ggcgacagcc
     6301 ggaactgccg tgccggtgtg cagattaatg acagcggtgc ggcgctggga tattacgtca
     6361 gcgaggacgg gtatcctggc tggatgccgc agaaatggac atggataccc cgtgagttac
     6421 ccggcgggcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     6481 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     6541 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     6601 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     6661 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     6721 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     6781 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     6841 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     6901 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     6961 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     7021 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     7081 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     7141 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     7201 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     7261 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     7321 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     7381 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     7441 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     7501 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     7561 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     7621 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     7681 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     7741 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     7801 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     7861 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     7921 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     7981 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     8041 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     8101 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     8161 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     8221 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     8281 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     8341 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     8401 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     8461 aatactcat
//
