LOCUS       sequence-347134-        8244 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TF B self-activation cassette in MultiFate-2.1 and MultiFate-2.3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8244)
  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 8244)
  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: 
            MF64_PB-TRE3G-6x42bs-6x(37bs_37bs)-miniCMV-NLS-FKBP12F36V-37ZFR2AR11
            AR39AR67A-VP16-NLS-DHFR-IRES-mCitrine-PEST-BGHpA
FEATURES             Location/Qualifiers
     source          1..8244
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(28..456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(138..159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     350..369
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     583..605
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     597..614
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     598..614
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   744..806
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    1260..1278
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1296..1314
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1332..1350
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1368..1386
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1404..1422
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1440..1458
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1822..1846
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1930..1939
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1942..1962
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1966..2286
                     /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             2809..2829
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2839..2862
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    join(3358..3932,3933..3935,3936..3944)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3525..3542)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3752..3771
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(4251..4270)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(4822..4839)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4828..5035
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5568..5602
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6093..6109)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6093..6109)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6106..6128)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6117..6133
                     /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(6141..6147,6148..6165,6166..6171))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6186..6207
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6324..6341)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6495..7083)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6575..6594)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7254..8045,8046..8114))
                     /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     7877..7896
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8115..8219)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
       61 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
      121 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
      241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      361 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca
      481 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
      541 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
      601 aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg gaggacgggc
      661 agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg ctcgacacgc
      721 tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg tacgttaaag
      781 ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag gtttatttat
      841 taatttgaat agatattaag ttttattata tttacactta catactaata ataaattcaa
      901 caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa aatttcttct
      961 ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag ggatgtaatt
     1021 acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg
     1081 ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc
     1141 acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg
     1201 gatacgggga aaaggcctcc acggccacta gtggcctcca cggccacaag cttcatatgt
     1261 ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata gagaacgtat
     1321 gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc ctatcagtga
     1381 tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct acagtttact
     1441 ccctatcagt gatagagaac gtatatccag tttactcccc gacgctgctg acgctgctga
     1501 cgctgctgac gctgctgacg ctgctgacgc tgctcgtcgc caattgtcgc cggcgtgagg
     1561 acgtgttgag gacgtgttct agagcctatt ttgaggacgt gttgaggacg tgtatcgata
     1621 tatttagtga ggacgtgttg aggacgtgtt cacgtactat ctatgaggac gtgttgagga
     1681 cgtgttatga cctaaggagt gaggacgtgt tgaggacgtg tacgtatata cgcgttgagg
     1741 acgtgttgag gacgtgtcct agaccggttg atagagaacg tataagcttt aggcgtgtac
     1801 ggtgggcgcc tataaaagca gagctcgttt agtgaaccgt cagatcgcct ggagcaattc
     1861 cacaacactt ttgtcttata ccaactttcc gtaccacttc ctaccctcgt aaatcgacga
     1921 agcttctagg ccaccatggg acccaagaaa aagcggaagg tgtccggagt gcaggtggag
     1981 actatctccc caggagacgg gcgcaccttc cccaagcgcg gccagacctg cgtggtgcac
     2041 tacaccggga tgcttgaaga tggaaagaaa gttgattcct cccgggacag aaacaagccc
     2101 tttaagttta tgctaggcaa gcaggaggtg atccgaggct gggaagaagg ggttgcccag
     2161 atgagtgtgg gtcagagagc caaactgact atatctccag attatgccta tggtgccact
     2221 gggcacccag gcatcatccc accacatgcc actctcgtct tcgatgtgga gcttctaaaa
     2281 ctggaaggat ccgaggcccc attccagtgc agaatctgca tggctaactt ctcccggaac
     2341 ttcatcctgc agagacacac cagaacacac acaggcgaga agcccttcca gtgccggatc
     2401 tgtatggcca acttttccga cagagccaac ctgcggagac acctgagaac ccataccggc
     2461 gaaaagcctt ttcagtgtcg catctgcatg gccaatttca gcaggcacga ccagctgaca
     2521 agacatctga aaacccacac tggcgagaaa ggcggcggat cctctggcgc tcctcctacc
     2581 gatgtgtctc tgggagatga gctgcacctg gacggcgagg atgtggctat ggctcatgcc
     2641 gacgctctgg acgacttcga cttggatatg ctcggcgacg gcgactctcc tggacctggc
     2701 tttacccctc atgactctgc cccttacggc gccctggata tggccgattt cgagttcgag
     2761 cagatgttca ccgatgctct gggcatcgat gagtacggcg gaggctcccc caagaaaaag
     2821 cggaaggtgg gctcgagcga ctacaaggac gacgacgaca agtcgagcat ctccctgatc
     2881 gctgccctgg ctgtggatta cgtgatcgga atggaaaacg ccatgccatg gaacctgcct
     2941 gctgacctgg cctggttcaa gcggaacacc ctgaacaagc ccgtgatcat gggcagacac
     3001 acctgggagt ctatcggcag acctctgcct ggccggaaga acatcatcct gtcctctcag
     3061 ccttccaccg acgacagagt gacctgggtc aagtctgtgg acgaggctat cgctgcttgt
     3121 ggcgacgtgc cagagatcat ggttatcggc ggaggcagag tgatcgagca gttcctgcct
     3181 aaggctcaga agctgtatct gacccacatc gacgccgaag tggaaggcga tacccacttt
     3241 cctgactacg agcccgacga ctgggagtcc gtgttctctg agttccacga cgccgatgct
     3301 cagaactccc actcctactg cttcgagatc ctggagcgga gataggtcga ccagatcccc
     3361 ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc
     3421 gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa
     3481 acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat
     3541 gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac
     3601 aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg
     3661 cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt
     3721 tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg
     3781 gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac
     3841 atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga
     3901 cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatggtg agcaagggcg
     3961 aggagctgtt caccggcgtg gtgcccattc tggtggaact ggatggggat gtgaacggcc
     4021 acaagttctc cgtgtctggc gaaggcgaag gggatgctac ctacggcaag ctgaccctga
     4081 agttcatctg caccaccgga aagctgcccg tgccttggcc tacactggtc accacctttg
     4141 gctacggcct gatgtgcttc gctagatacc ccgaccatat gaagcaacac gacttcttca
     4201 agtccgccat gcctgagggc tacgtgcaag agcggaccat cttctttaag gacgacggca
     4261 actacaagac cagggccgaa gtgaagttcg agggcgacac cctggtcaac cggatcgagc
     4321 tgaagggcat cgacttcaaa gaggacggca acatcctggg ccacaagctc gagtacaact
     4381 acaactccca caacgtgtac atcatggccg acaagcagaa aaacggcatc aaagtgaact
     4441 tcaagatcag gcacaacatc gaggacggct ccgtgcagct ggctgatcac taccagcaga
     4501 acacccctat cggcgacgga cctgtgctgc tgcctgataa ccactacctg tcctaccagt
     4561 ccaagctgag caaggacccc aacgagaagc gggaccacat ggtgctgctg gaatttgtga
     4621 ccgccgctgg catcaccctc ggcatggatg agctgtacaa gtccggcaag cttagccatg
     4681 gcttcccgcc ggaggtggag gagcaggatg atggcacgct gcccatgtct tgtgcccagg
     4741 agagcgggat ggaccgtcac cctgcagcct gtgcttctgc taggatcaat gtgtgagcta
     4801 gcgaattcga atttaaatcg gcctcgactg tgccttctag ttgccagcca tctgttgttt
     4861 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     4921 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     4981 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggacgcgg
     5041 ccgcgggccc attggtatgg ctttttcccc gtatcccccc aggtgtctgc aggctcaaag
     5101 agcagcgaga agcgttcaga ggaaagcgat cccgtgccac cttccccgtg cccgggctgt
     5161 ccccgcacgc tgccggctcg gggatgcggg gggagcgccg gaccggagcg gagccccggg
     5221 cggctcgctg ctgcccccta gcgggggagg gacgtaatta catccctggg ggctttgggg
     5281 gggggctgtc cctgatatct ataacaagaa aatatatata taataagtta tcacgtaagt
     5341 agaacatgaa ataacaatat aattatcgta tgagttaaat cttaaaagtc acgtaaaaga
     5401 taatcatgcg tcattttgac tcacgcggtc gttatagttc aaaatcagtg acacttaccg
     5461 cattgacaag cacgcctcac gggagctcca agcggcgact gagatgtcct aaatgcacag
     5521 cgacggattc gcgctattta gaaagagaga gcaatatttc aagaatgcat gcgtcaattt
     5581 tacgcagact atctttctag ggttaatcta gctgcatcag gatcatatcg tcgggtcttt
     5641 tttccggctc agtcatcgcc caagctggcg ctatctgggc atcggggagg aagaagcccg
     5701 tgccttttcc cgcgaggttg aagcggcatg gaaagagttt gccgaggatg actgctgctg
     5761 cattgacgtt gagcgaaaac gcacgtttac catgatgatt cgggaaggtg tggccatgca
     5821 cgcctttaac ggtgaactgt tcgttcaggc cacctgggat accagttcgt cgcggctttt
     5881 ccggacacag ttccggatgg tcagcccgaa gcgcatcagc aacccgaaca ataccggcga
     5941 cagccggaac tgccgtgccg gtgtgcagat taatgacagc ggtgcggcgc tgggatatta
     6001 cgtcagcgag gacgggtatc ctggctggat gccgcagaaa tggacatgga taccccgtga
     6061 gttacccggc gggcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     6121 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     6181 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     6241 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     6301 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     6361 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     6421 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     6481 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     6541 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     6601 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     6661 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     6721 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6781 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6841 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6901 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6961 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     7021 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     7081 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     7141 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     7201 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     7261 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     7321 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     7381 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     7441 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     7501 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     7561 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     7621 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     7681 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     7741 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7801 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7861 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7921 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7981 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     8041 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     8101 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     8161 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     8221 acatttcccc gaaaagtgcc acct
//
