LOCUS       sequence-347271-        8325 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TF A self-activation cassette in MultiFate-2.3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8325)
  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 8325)
  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: 
            MF84_PB-14xUAS-6x42bs-6x(BCRbs_BCRbs)-miniCMV-NLS-FKBP12F36V-BCRZFR3
            9A-VP16-NLS-DHFR-IRES-mCherry-PEST-BGHpA
FEATURES             Location/Qualifiers
     source          1..8325
                     /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     2042..2066
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2150..2159
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2162..2182
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2186..2506
                     /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             3029..3049
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3059..3082
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    join(3578..4152,4153..4155,4156..4164)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3745..3762)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3972..3991
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4165..4872
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4310..4328)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4578..4597
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(5033..5050)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5039..5246
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5779..5813
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6304..6320)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6304..6320)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6317..6339)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6328..6344
                     /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(6352..6358,6359..6376,6377..6382))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6397..6418
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6535..6552)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6706..7294)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6786..6805)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7465..8256,8257..8325))
                     /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     8088..8107
                     /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 aggcggagta
     1381 ctgtcctccg ggctggcgga gtactgtcct ccggcaaggt cggagtactg tcctccgaca
     1441 ctagaggtcg gagtactgtc ctccgacgca aggcggagta ctgtcctccg ggctgcggag
     1501 tactgtcctc cggcaaggtc ggagtactgt cctccgacac tagaggtcgg agtactgtcc
     1561 tccgacgcaa ggtcggagta ctgtcctccg acactagagg tcggagtact gtcctccgac
     1621 gcaaggtcgg agtactgtcc tccgacacta gaggtcggag tactgtcctc cgacgcaagg
     1681 cggagtactg tcctccgggc tggcggagta ctgtcctccg gcaagggctc gacgctgctg
     1741 acgctgctga cgctgctgac gctgctgacg ctgctgacgc tgctcaattg tcgccggcgg
     1801 cagaagccgc agaagcctct agagcctatt tgcagaagcc gcagaagcca tcgatatatt
     1861 taggcagaag ccgcagaagc ctcacgtact atctagcaga agccgcagaa gcctatgacc
     1921 taaggaggca gaagccgcag aagccacgta tatacgcgtg cagaagccgc agaagcccct
     1981 agaccggttg atagagaacg tataagcttt aggcgtgtac ggtgggcgcc tataaaagca
     2041 gagctcgttt agtgaaccgt cagatcgcct ggagcaattc cacaacactt ttgtcttata
     2101 ccaactttcc gtaccacttc ctaccctcgt aaatcgacga agcttctagg ccaccatggg
     2161 acccaagaaa aagcggaagg tgtccggagt gcaggtggag actatctccc caggagacgg
     2221 gcgcaccttc cccaagcgcg gccagacctg cgtggtgcac tacaccggga tgcttgaaga
     2281 tggaaagaaa gttgattcct cccgggacag aaacaagccc tttaagttta tgctaggcaa
     2341 gcaggaggtg atccgaggct gggaagaagg ggttgcccag atgagtgtgg gtcagagagc
     2401 caaactgact atatctccag attatgccta tggtgccact gggcacccag gcatcatccc
     2461 accacatgcc actctcgtct tcgatgtgga gcttctaaaa ctggaaggat ccgagcggcc
     2521 attccagtgc agaatctgca tgcggaactt ctccgactct cccacactgc ggagacacac
     2581 cagaacacac acaggcgaga agcccttcca gtgccggatc tgtatggcca acttttccca
     2641 gggcgccaac ctgagaaggc acctgagaac ccataccggc gaaaagcctt ttcagtgtcg
     2701 catctgcatg agaaacttta gccaggctaa caccctgcag cgccatctga aaacccacac
     2761 tggcgagaaa ggaggcggat cctctggcgc tcctcctacc gatgtgtctc tgggagatga
     2821 gctgcacctg gacggcgagg atgtggctat ggctcatgcc gacgctctgg acgacttcga
     2881 cttggatatg ctcggcgacg gcgactctcc tggacctggc tttacccctc atgactctgc
     2941 cccttacggc gccctggata tggccgattt cgagttcgag cagatgttca ccgatgctct
     3001 gggcatcgat gagtacggcg gaggctcccc caagaaaaag cggaaggtgg gctcgagcga
     3061 ctacaaggac gacgacgaca agtcgagcat ctccctgatc gctgccctgg ctgtggatta
     3121 cgtgatcgga atggaaaacg ccatgccatg gaacctgcct gctgacctgg cctggttcaa
     3181 gcggaacacc ctgaacaagc ccgtgatcat gggcagacac acctgggagt ctatcggcag
     3241 acctctgcct ggccggaaga acatcatcct gtcctctcag ccttccaccg acgacagagt
     3301 gacctgggtc aagtctgtgg acgaggctat cgctgcttgt ggcgacgtgc cagagatcat
     3361 ggttatcggc ggaggcagag tgatcgagca gttcctgcct aaggctcaga agctgtatct
     3421 gacccacatc gacgccgaag tggaaggcga tacccacttt cctgactacg agcccgacga
     3481 ctgggagtcc gtgttctctg agttccacga cgccgatgct cagaactccc actcctactg
     3541 cttcgagatc ctggagcgga gataggtcga ccagatcccc ctctccctcc ccccccccta
     3601 acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt
     3661 ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga
     3721 cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg
     3781 tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt
     3841 gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat
     3901 aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg
     3961 aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg
     4021 taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt
     4081 cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa
     4141 cacgatgata atatggccac aaccatggtg agcaagggcg aggaggataa catggccatc
     4201 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc
     4261 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     4321 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac
     4381 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
     4441 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
     4501 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
     4561 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc
     4621 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg
     4681 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg
     4741 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac
     4801 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggat
     4861 gagctgtaca agtccggcaa gcttagccat ggcttcccgc cggaggtgga ggagcaggat
     4921 gatggcacgc tgcccatgtc ttgtgcccag gagagcggga tggaccgtca ccctgcagcc
     4981 tgtgcttctg ctaggatcaa tgtgtgagct agcgaattcg aatttaaatc ggcctcgact
     5041 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     5101 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     5161 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     5221 gaagacaata gcaggcatgc tggggacgcg gccgcgggcc cattggtatg gctttttccc
     5281 cgtatccccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga
     5341 tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc ggggatgcgg
     5401 ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag
     5461 ggacgtaatt acatccctgg gggctttggg ggggggctgt ccctgatatc tataacaaga
     5521 aaatatatat ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt
     5581 atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt
     5641 cgttatagtt caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc
     5701 aagcggcgac tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag
     5761 agcaatattt caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaatct
     5821 agctgcatca ggatcatatc gtcgggtctt ttttccggct cagtcatcgc ccaagctggc
     5881 gctatctggg catcggggag gaagaagccc gtgccttttc ccgcgaggtt gaagcggcat
     5941 ggaaagagtt tgccgaggat gactgctgct gcattgacgt tgagcgaaaa cgcacgttta
     6001 ccatgatgat tcgggaaggt gtggccatgc acgcctttaa cggtgaactg ttcgttcagg
     6061 ccacctggga taccagttcg tcgcggcttt tccggacaca gttccggatg gtcagcccga
     6121 agcgcatcag caacccgaac aataccggcg acagccggaa ctgccgtgcc ggtgtgcaga
     6181 ttaatgacag cggtgcggcg ctgggatatt acgtcagcga ggacgggtat cctggctgga
     6241 tgccgcagaa atggacatgg ataccccgtg agttacccgg cgggcgcgct tggcgtaatc
     6301 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     6361 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     6421 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     6481 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     6541 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     6601 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     6661 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     6721 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     6781 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     6841 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     6901 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     6961 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     7021 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     7081 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     7141 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     7201 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     7261 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     7321 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     7381 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     7441 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     7501 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     7561 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     7621 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     7681 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     7741 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     7801 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     7861 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     7921 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     7981 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     8041 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     8101 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     8161 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     8221 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     8281 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcat
//
