LOCUS       sequence-119433-        6552 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SKARS: Fus3 and Kss1 MAPK activity sensor: Ste7DS 2xNLS(4A) mCherry 
            Non functional sensor with non-phosphorylatable NLS.
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: pED53 pRS306 pRPS2-Ste7DS NLS(4A)-mCherry
FEATURES             Location/Qualifiers
     source          1..6552
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1309..2010
                     /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(1448..1466)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1716..1735
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      2026..2273
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2042..2060)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(2296..2314)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2335..2351)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2335..2351)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2348..2370)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2359..2375
                     /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(2383..2389,2390..2407,2408..2413))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(2566..2583)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2737..3325)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(3496..4287,4288..4356))
                     /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     4119..4138
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4357..4461)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4529..4547
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4585..4607)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4707..4726
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4746..4966
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             4967..5770
                     /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(5899..6354)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6036..6057)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6248..6267
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6484..6506
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6498..6515
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6499..6515
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6522..6541
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6522..6540
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 tcgttgccac gattggaata ccctcaaaat tcccactttt actatgcttc aattgaccct
       61 tcggtatctc agcatctagg tagctttcat tgagattcat agttccgatg tactgattct
      121 acgtttgtgt actttgatag acttttttat tcaattcgta ttcactacta tctttgtctc
      181 gtaccgcggt ttttatcctt tatttcagcc ctacgagata gaacgatgga gcctagtaat
      241 accattttat gtaagcaaaa taagaagaaa gactaaaaga attaggatat gttgttaatc
      301 agggataaag gtacgcatac tagccttact acgtaaatat aaggttacgg tttacgatca
      361 ctattgcaag atactattgt ttagcattgg caaagacttg acgggaaatc tttgattggg
      421 cccaaccata ttcattaaag atccgggatg tgagcttgaa ctgcttttcg acgcgaagaa
      481 ggtttacacg tattttagta attggcttat tcactaagga ttcttaaggt tttcttaata
      541 gttttctacg tcggcatgcg attgtttggt ttagaagact gctttctaaa tatggttggg
      601 tgtatttaag ctagacccat acacccgctc tatgggatta tttacttgtt tgaattttaa
      661 gatttgtgat aatggaactg gacgcaaaca tttgatggaa aacgcatgtc atcattaacg
      721 aggtaacgta ggtatctgtc ctgccttagt attgcacgca gcttcccagg acgcctagct
      781 attttttcat ctattcccct ctgtagtaac gtaagagttt tcaagttttt aattcagact
      841 ttctcttcct ttgtttccaa tttccttcct tactgcttga taccttttca atcccaaaga
      901 aaccgtgttc tttatatatt gtcgattgaa agttacctac atcaactttc cgtgttccat
      961 tccgactata acaaacaacc aataagctca actaattaag taaagcttat gtttcaacga
     1021 aagactttac agagaaggaa cttgaaaggg ctcaatctta acctgcaccc agatgtgggc
     1081 aataatggcc aattgcagga aaagacatct agacaacaaa tgggtcgtgg ttcggaattt
     1141 gaagaactag gtgccccgct aaaaaaacta aaaatagcac cagatacggc ttctggtttg
     1201 gttgaattcc aacaaatggg tcgtggttcg gaatttgaag aactaggtgc cccgctaaaa
     1261 aaactaaaaa tagcaccaga tacggcttct ggtttggtta gatctgctag caagggcgag
     1321 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     1381 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     1441 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     1501 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     1561 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     1621 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
     1681 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     1741 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     1801 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     1861 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     1921 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     1981 tccaccggcg gcatggacga gctgtacaag ggatcttaac tcgagtcatg taattagtta
     2041 tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta
     2101 gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt
     2161 tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta
     2221 tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcggccggt
     2281 acccagcttt tgttcccttt agtgagggtt aattccgagc ttggcgtaat catggtcata
     2341 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatag gagccggaag
     2401 cataaagtgt aaagcctggg gtgcctaatg agtgaggtaa ctcacattaa ttgcgttgcg
     2461 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     2521 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     2581 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2641 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2701 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcg gcccccctga
     2761 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2821 ataccaggcg ttcccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2881 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     2941 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3001 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3061 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3121 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     3181 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3241 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3301 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3361 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3421 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3481 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3541 atttcgttca tccatagttg cctgactgcc cgtcgtgtag ataactacga tacgggaggg
     3601 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     3661 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3721 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3781 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3841 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3901 gttgtgaaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3961 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4021 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4081 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4141 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4201 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4261 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4321 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4381 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4441 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     4501 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     4561 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     4621 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     4681 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     4741 cgcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc ggtttctttg
     4801 aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg agcacagact
     4861 tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc cagtattctt
     4921 aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt cgaaagctac
     4981 atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat ttaatatcat
     5041 gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca aggaattact
     5101 ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg tggatatctt
     5161 gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg ccaagtacaa
     5221 ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca aattgcagta
     5281 ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac acggtgtggt
     5341 gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa aggaacctag
     5401 aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg gagaatatac
     5461 taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct ttattgctca
     5521 aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac ccggtgtggg
     5581 tttagatgac aagggagacg cattgggtca acagtataga accgtggatg atgtggtctc
     5641 tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa gggatgctaa
     5701 ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa gatgcggcca
     5761 gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac aaattagagc
     5821 ttcaatttaa ttatatcagt tattaccctg cggtgtgaaa taccgcacag atgcgtaagg
     5881 agaaaatacc gcatcaggaa attgtaaacg ttaatatttt gttaaaattc gcgttaaatt
     5941 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     6001 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     6061 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     6121 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     6181 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     6241 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     6301 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcgcgcc
     6361 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     6421 tacgccagct ggcgaagggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     6481 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac tcactatagg
     6541 gcgaattgga gc
//
