LOCUS       sequence-119429-        6552 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SKARS: Fus3 and Kss1 MAPK activity sensor: Ste7DS 2xNLS mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6552)
  AUTHORS   Durandau E, Aymoz D, Pelet S
  TITLE     Dynamic single cell measurements of kinase activity by synthetic 
            kinase activity relocation sensors.
  JOURNAL   BMC Biol. 2015 Aug 1;13:55. doi: 10.1186/s12915-015-0163-z.
  PUBMED    26231587
REFERENCE   2  (bases 1 to 6552)
  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: "BMC Biol.";
            date: "2015-08-1"; volume: "13:55. doi"; pages: " 
            10.1186/s12915-015-0163-z"
COMMENT     Sequence Label: pED45: pRS306 pRPS2-Ste7DS-NLS-mCherry
FEATURES             Location/Qualifiers
     source          1..6552
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     104..126
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     118..135
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     119..135
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     142..161
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        142..160
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1481..2182
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="SKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQ
                     TAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFED
                     GGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGE
                     IKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEG
                     RHSTGGMDELYK"
     primer_bind     complement(1620..1638)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1888..1907
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      2198..2445
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2214..2232)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(2468..2486)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2507..2523)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2507..2523)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2520..2542)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2531..2547
                     /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(2555..2561,2562..2579,2580..2585))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(2738..2755)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2909..3497)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(3668..4459,4460..4528))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4291..4310
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4529..4633)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4701..4719
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4757..4779)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4879..4898
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4918..5138
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             5139..5942
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(6071..6526)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6208..6229)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6420..6439
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaagggg
       61 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
      121 aaaacgacgg ccagtgaatt gtaatacgac tcactatagg gcgaattgga gctcgttgcc
      181 acgattggaa taccctcaaa attcccactt ttactatgct tcaattgacc cttcggtatc
      241 tcagcatcta ggtagctttc attgagattc atagttccga tgtactgatt ctacgtttgt
      301 gtactttgat agactttttt attcaattcg tattcactac tatctttgtc tcgtaccgcg
      361 gtttttatcc tttatttcag ccctacgaga tagaacgatg gagcctagta ataccatttt
      421 atgtaagcaa aataagaaga aagactaaaa gaattaggat atgttgttaa tcagggataa
      481 aggtacgcat actagcctta ctacgtaaat ataaggttac ggtttacgat cactattgca
      541 agatactatt gtttagcatt ggcaaagact tgacgggaaa tctttgattg ggcccaacca
      601 tattcattaa agatccggga tgtgagcttg aactgctttt cgacgcgaag aaggtttaca
      661 cgtattttag taattggctt attcactaag gattcttaag gttttcttaa tagttttcta
      721 cgtcggcatg cgattgtttg gtttagaaga ctgctttcta aatatggttg ggtgtattta
      781 agctagaccc atacacccgc tctatgggat tatttacttg tttgaatttt aagatttgtg
      841 ataatggaac tggacgcaaa catttgatgg aaaacgcatg tcatcattaa cgaggtaacg
      901 taggtatctg tcctgcctta gtattgcacg cagcttccca ggacgcctag ctattttttc
      961 atctattccc ctctgtagta acgtaagagt tttcaagttt ttaattcaga ctttctcttc
     1021 ctttgtttcc aatttccttc cttactgctt gatacctttt caatcccaaa gaaaccgtgt
     1081 tctttatata ttgtcgattg aaagttacct acatcaactt tccgtgttcc attccgacta
     1141 taacaaacaa ccaataagct caactaatta agtaaagctt atgtttcaac gaaagacttt
     1201 acagagaagg aacttgaaag ggctcaatct taacctgcac ccagatgtgg gcaataatgg
     1261 ccaattgcag gaaaagacat ctagacaaca aatgggtcgt ggttcggaat ttgaagaact
     1321 aggttccccg ctaaaaaaac taaaaatatc accagatacg gcttctggtt tggttgaatt
     1381 ccaacaaatg ggtcgtggtt cggaatttga agaactaggt tccccgctaa aaaaactaaa
     1441 aatatcacca gatacggctt ctggtttggt tagatctgct agcaagggcg aggaggataa
     1501 catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg
     1561 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     1621 caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca
     1681 gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
     1741 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
     1801 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
     1861 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
     1921 ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag
     1981 gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa
     2041 gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca
     2101 caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg
     2161 cggcatggac gagctgtaca agggatctta actcgagtca tgtaattagt tatgtcacgc
     2221 ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct
     2281 gaagtctagg tccctattta tttttttata gttatgttag tattaagaac gttatttata
     2341 tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa
     2401 accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcggccg gtacccagct
     2461 tttgttccct ttagtgaggg ttaattccga gcttggcgta atcatggtca tagctgtttc
     2521 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat aggagccgga agcataaagt
     2581 gtaaagcctg gggtgcctaa tgagtgaggt aactcacatt aattgcgttg cgctcactgc
     2641 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     2701 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     2761 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     2821 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     2881 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct cggcccccct gacgagcatc
     2941 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3001 cgttcccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3061 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt
     3121 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3181 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3241 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3301 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     3361 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3421 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3481 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3541 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     3601 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3661 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3721 catccatagt tgcctgactg cccgtcgtgt agataactac gatacgggag ggcttaccat
     3781 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3841 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3901 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3961 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4021 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgaa
     4081 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4141 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4201 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4261 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4321 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4381 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     4441 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     4501 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     4561 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     4621 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     4681 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     4741 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     4801 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     4861 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac cacgcttttc
     4921 aattcaattc atcatttttt ttttattctt ttttttgatt tcggtttctt tgaaattttt
     4981 ttgattcggt aatctccgaa cagaaggaag aacgaaggaa ggagcacaga cttagattgg
     5041 tatatatacg catatgtagt gttgaagaaa catgaaattg cccagtattc ttaacccaac
     5101 tgcacagaac aaaaacctgc aggaaacgaa gataaatcat gtcgaaagct acatataagg
     5161 aacgtgctgc tactcatcct agtcctgttg ctgccaagct atttaatatc atgcacgaaa
     5221 agcaaacaaa cttgtgtgct tcattggatg ttcgtaccac caaggaatta ctggagttag
     5281 ttgaagcatt aggtcccaaa atttgtttac taaaaacaca tgtggatatc ttgactgatt
     5341 tttccatgga gggcacagtt aagccgctaa aggcattatc cgccaagtac aattttttac
     5401 tcttcgaaga cagaaaattt gctgacattg gtaatacagt caaattgcag tactctgcgg
     5461 gtgtatacag aatagcagaa tgggcagaca ttacgaatgc acacggtgtg gtgggcccag
     5521 gtattgttag cggtttgaag caggcggcag aagaagtaac aaaggaacct agaggccttt
     5581 tgatgttagc agaattgtca tgcaagggct ccctatctac tggagaatat actaagggta
     5641 ctgttgacat tgcgaagagc gacaaagatt ttgttatcgg ctttattgct caaagagaca
     5701 tgggtggaag agatgaaggt tacgattggt tgattatgac acccggtgtg ggtttagatg
     5761 acaagggaga cgcattgggt caacagtata gaaccgtgga tgatgtggtc tctacaggat
     5821 ctgacattat tattgttgga agaggactat ttgcaaaggg aagggatgct aaggtagagg
     5881 gtgaacgtta cagaaaagca ggctgggaag catatttgag aagatgcggc cagcaaaact
     5941 aaaaaactgt attataagta aatgcatgta tactaaactc acaaattaga gcttcaattt
     6001 aattatatca gttattaccc tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     6061 ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     6121 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     6181 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     6241 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     6301 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     6361 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     6421 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     6481 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcgcg ccattcgcca
     6541 ttcaggctgc gc
//
