LOCUS       sequence-347070-        8327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TF B self-activation cassette in MultiFate-2.2 and MultiFate-3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8327)
  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 8327)
  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: 
            MF83_PB-TRE3G-6x42bs-10x(37bs_37bs)-miniCMV-NLS-FKBP12F36V-37ZFR2AR1
            1AR39AR67A-VP16-NLS-DHFR-IRES-mCitrine-PEST-BGHpA
FEATURES             Location/Qualifiers
     source          1..8327
                     /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)"
     protein_bind    1390..1408
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1426..1444
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1462..1480
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1498..1516
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1534..1552
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1570..1588
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2035..2059
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2143..2152
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2155..2175
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2179..2499
                     /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             3022..3042
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3052..3075
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    join(3571..4145,4146..4148,4149..4157)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3738..3755)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3965..3984
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(4464..4483)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(5035..5052)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5041..5248
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5781..5815
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6306..6322)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6306..6322)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6319..6341)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6330..6346
                     /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(6354..6360,6361..6378,6379..6384))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6399..6420
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6537..6554)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6708..7296)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6788..6807)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7467..8258,8259..8327))
                     /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     8090..8109
                     /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 cttcatatgt ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata
     1441 gagaacgtat gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc
     1501 ctatcagtga tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct
     1561 acagtttact ccctatcagt gatagagaac gtaggtccag tttactcccg acgctgctga
     1621 cgctgctgac gctgctgacg ctgctgacgc tgctgacgct gctcaattgt cgccggcgtc
     1681 tgaggacgtg ttgaggacgt gttcacgtac tgaggacgtg ttgaggacgt gttatgacct
     1741 tgaggacgtg ttgaggacgt gttagagctg aggacgtgtt gaggacgtgt atcgatattg
     1801 aggacgtgtt gaggacgtgt tctagagctg aggacgtgtt gaggacgtgt atcgatattg
     1861 aggacgtgtt gaggacgtgt tcacgtactg aggacgtgtt gaggacgtgt tatgaccttg
     1921 aggacgtgtt gaggacgtgt acgtaggttg aggacgtgtt gaggacgtgt cctagaccgg
     1981 ttgatagaga acgtataagc tttaggcgtg tacggtgggc gcctataaaa gcagagctcg
     2041 tttagtgaac cgtcagatcg cctggagcaa ttccacaaca cttttgtctt ataccaactt
     2101 tccgtaccac ttcctaccct cgtaaatcga cgaagcttct aggccaccat gggacccaag
     2161 aaaaagcgga aggtgtccgg agtgcaggtg gagactatct ccccaggaga cgggcgcacc
     2221 ttccccaagc gcggccagac ctgcgtggtg cactacaccg ggatgcttga agatggaaag
     2281 aaagttgatt cctcccggga cagaaacaag ccctttaagt ttatgctagg caagcaggag
     2341 gtgatccgag gctgggaaga aggggttgcc cagatgagtg tgggtcagag agccaaactg
     2401 actatatctc cagattatgc ctatggtgcc actgggcacc caggcatcat cccaccacat
     2461 gccactctcg tcttcgatgt ggagcttcta aaactggaag gatccgaggc cccattccag
     2521 tgcagaatct gcatggctaa cttctcccgg aacttcatcc tgcagagaca caccagaaca
     2581 cacacaggcg agaagccctt ccagtgccgg atctgtatgg ccaacttttc cgacagagcc
     2641 aacctgcgga gacacctgag aacccatacc ggcgaaaagc cttttcagtg tcgcatctgc
     2701 atggccaatt tcagcaggca cgaccagctg acaagacatc tgaaaaccca cactggcgag
     2761 aaaggcggcg gatcctctgg cgctcctcct accgatgtgt ctctgggaga tgagctgcac
     2821 ctggacggcg aggatgtggc tatggctcat gccgacgctc tggacgactt cgacttggat
     2881 atgctcggcg acggcgactc tcctggacct ggctttaccc ctcatgactc tgccccttac
     2941 ggcgccctgg atatggccga tttcgagttc gagcagatgt tcaccgatgc tctgggcatc
     3001 gatgagtacg gcggaggctc ccccaagaaa aagcggaagg tgggctcgag cgactacaag
     3061 gacgacgacg acaagtcgag catctccctg atcgctgccc tggctgtgga ttacgtgatc
     3121 ggaatggaaa acgccatgcc atggaacctg cctgctgacc tggcctggtt caagcggaac
     3181 accctgaaca agcccgtgat catgggcaga cacacctggg agtctatcgg cagacctctg
     3241 cctggccgga agaacatcat cctgtcctct cagccttcca ccgacgacag agtgacctgg
     3301 gtcaagtctg tggacgaggc tatcgctgct tgtggcgacg tgccagagat catggttatc
     3361 ggcggaggca gagtgatcga gcagttcctg cctaaggctc agaagctgta tctgacccac
     3421 atcgacgccg aagtggaagg cgatacccac tttcctgact acgagcccga cgactgggag
     3481 tccgtgttct ctgagttcca cgacgccgat gctcagaact cccactccta ctgcttcgag
     3541 atcctggagc ggagataggt cgaccagatc cccctctccc tccccccccc ctaacgttac
     3601 tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat
     3661 attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat
     3721 tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga
     3781 agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca
     3841 gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac
     3901 acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt
     3961 caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca
     4021 ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt
     4081 aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg
     4141 ataatatggc cacaaccatg gtgagcaagg gcgaggagct gttcaccggc gtggtgccca
     4201 ttctggtgga actggatggg gatgtgaacg gccacaagtt ctccgtgtct ggcgaaggcg
     4261 aaggggatgc tacctacggc aagctgaccc tgaagttcat ctgcaccacc ggaaagctgc
     4321 ccgtgccttg gcctacactg gtcaccacct ttggctacgg cctgatgtgc ttcgctagat
     4381 accccgacca tatgaagcaa cacgacttct tcaagtccgc catgcctgag ggctacgtgc
     4441 aagagcggac catcttcttt aaggacgacg gcaactacaa gaccagggcc gaagtgaagt
     4501 tcgagggcga caccctggtc aaccggatcg agctgaaggg catcgacttc aaagaggacg
     4561 gcaacatcct gggccacaag ctcgagtaca actacaactc ccacaacgtg tacatcatgg
     4621 ccgacaagca gaaaaacggc atcaaagtga acttcaagat caggcacaac atcgaggacg
     4681 gctccgtgca gctggctgat cactaccagc agaacacccc tatcggcgac ggacctgtgc
     4741 tgctgcctga taaccactac ctgtcctacc agtccaagct gagcaaggac cccaacgaga
     4801 agcgggacca catggtgctg ctggaatttg tgaccgccgc tggcatcacc ctcggcatgg
     4861 atgagctgta caagtccggc aagcttagcc atggcttccc gccggaggtg gaggagcagg
     4921 atgatggcac gctgcccatg tcttgtgccc aggagagcgg gatggaccgt caccctgcag
     4981 cctgtgcttc tgctaggatc aatgtgtgag ctagcgaatt cgaatttaaa tcggcctcga
     5041 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     5101 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     5161 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     5221 gggaagacaa tagcaggcat gctggggacg cggccgcggg cccattggta tggctttttc
     5281 cccgtatccc cccaggtgtc tgcaggctca aagagcagcg agaagcgttc agaggaaagc
     5341 gatcccgtgc caccttcccc gtgcccgggc tgtccccgca cgctgccggc tcggggatgc
     5401 ggggggagcg ccggaccgga gcggagcccc gggcggctcg ctgctgcccc ctagcggggg
     5461 agggacgtaa ttacatccct gggggctttg ggggggggct gtccctgata tctataacaa
     5521 gaaaatatat atataataag ttatcacgta agtagaacat gaaataacaa tataattatc
     5581 gtatgagtta aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt gactcacgcg
     5641 gtcgttatag ttcaaaatca gtgacactta ccgcattgac aagcacgcct cacgggagct
     5701 ccaagcggcg actgagatgt cctaaatgca cagcgacgga ttcgcgctat ttagaaagag
     5761 agagcaatat ttcaagaatg catgcgtcaa ttttacgcag actatctttc tagggttaat
     5821 ctagctgcat caggatcata tcgtcgggtc ttttttccgg ctcagtcatc gcccaagctg
     5881 gcgctatctg ggcatcgggg aggaagaagc ccgtgccttt tcccgcgagg ttgaagcggc
     5941 atggaaagag tttgccgagg atgactgctg ctgcattgac gttgagcgaa aacgcacgtt
     6001 taccatgatg attcgggaag gtgtggccat gcacgccttt aacggtgaac tgttcgttca
     6061 ggccacctgg gataccagtt cgtcgcggct tttccggaca cagttccgga tggtcagccc
     6121 gaagcgcatc agcaacccga acaataccgg cgacagccgg aactgccgtg ccggtgtgca
     6181 gattaatgac agcggtgcgg cgctgggata ttacgtcagc gaggacgggt atcctggctg
     6241 gatgccgcag aaatggacat ggataccccg tgagttaccc ggcgggcgcg cttggcgtaa
     6301 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     6361 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     6421 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     6481 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     6541 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     6601 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     6661 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6721 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6781 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     6841 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6901 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6961 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     7021 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     7081 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     7141 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     7201 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     7261 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     7321 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     7381 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     7441 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     7501 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     7561 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     7621 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7681 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7741 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7801 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7861 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7921 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7981 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     8041 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     8101 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     8161 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     8221 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     8281 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcat
//
