LOCUS       sequence-119436-        6455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SKARS: Fus3 and Kss1 MAPK activity sensor: Ste7DS 2xNLS mCitrine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6455)
  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 6455)
  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: pED73 pRS304 pRPS2-SteDS-NLS-mCitrine
FEATURES             Location/Qualifiers
     source          1..6455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        185..465
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             466..1140
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     rep_origin      complement(1237..1692)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1374..1395)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1586..1605
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1822..1844
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1836..1853
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1837..1853
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1860..1879
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1860..1878
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3199..3909
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLKEF
                     VTAAGITHGMDELYK"
     terminator      3925..4172
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3941..3959)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(4195..4213)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4234..4250)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4234..4250)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4247..4269)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4258..4274
                     /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(4282..4288,4289..4306,4307..4312))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4465..4482)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4636..5224)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5395..6186,6187..6255))
                     /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     6018..6037
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6256..6360)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6428..6446
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accaaacgac attactatat atataatata ggaagcattt aatagacagc atcgtaatat
      241 atgtgtactt tgcagttatg acgccagatg gcagtagtgg aagatattct ttattgaaaa
      301 atagcttgtc accttacgta caatcttgat ccggagcttt tctttttttg ccgattaaga
      361 attaattcgg tcgaaaaaag aaaaggagag ggccaagagg gagggcattg gtgactattg
      421 agcacgtgag tatacgtgat taagcacaca aaggcagctt ggagtatgtc tgttattaat
      481 ttcacaggta gttctggtcc attggtgaaa gtttgcggct tgcagagcac agaggccgca
      541 gaatgtgctc tagattccga tgctgacttg ctgggtatta tatgtgtgcc caatagaaag
      601 agaacaattg acccggttat tgcaaggaaa atttcaagtc ttgtaaaagc atataaaaat
      661 agttcaggca ctccgaaata cttggttggc gtgtttcgta atcaacctaa ggaggatgtt
      721 ttggctctgg tcaatgatta cggcattgat atcgtccaac tgcatggaga tgagtcgtgg
      781 caagaatacc aagagttcct cggtttgcca gttattaaaa gactcgtatt tccaaaagac
      841 tgcaacatac tactcagtgc agcttcacag aaacctcatt cgtttattcc cttgtttgat
      901 tcagaagcag gtgggacagg tgaacttttg gattggaact cgatttctga ctgggttgga
      961 aggcaagaga gccccgaaag cttacatttt atgttagctg gtggactgac gccagaaaat
     1021 gttggtgatg cgcttagatt aaatggcgtt attggtgttg atgtaagcgg aggtgtggag
     1081 acaaatggtg taaaagactc taacaaaata gcaaatttcg tcaaaaatgc taagaaatag
     1141 gttattactg agtagtattt atttaagtat tgtttgtgca cttgcctgcg gtgtgaaata
     1201 ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaaacgtt aatattttgt
     1261 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     1321 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     1381 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     1441 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     1501 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     1561 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     1621 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     1681 tacagggcgc gtcgcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     1741 tgcgggcctc ttcgctatta cgccagctgg cgaagggggg atgtgctgca aggcgattaa
     1801 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattgt
     1861 aatacgactc actatagggc gaattggagc tcgttgccac gattggaata ccctcaaaat
     1921 tcccactttt actatgcttc aattgaccct tcggtatctc agcatctagg tagctttcat
     1981 tgagattcat agttccgatg tactgattct acgtttgtgt actttgatag acttttttat
     2041 tcaattcgta ttcactacta tctttgtctc gtaccgcggt ttttatcctt tatttcagcc
     2101 ctacgagata gaacgatgga gcctagtaat accattttat gtaagcaaaa taagaagaaa
     2161 gactaaaaga attaggatat gttgttaatc agggataaag gtacgcatac tagccttact
     2221 acgtaaatat aaggttacgg tttacgatca ctattgcaag atactattgt ttagcattgg
     2281 caaagacttg acgggaaatc tttgattggg cccaaccata ttcattaaag atccgggatg
     2341 tgagcttgaa ctgcttttcg acgcgaagaa ggtttacacg tattttagta attggcttat
     2401 tcactaagga ttcttaaggt tttcttaata gttttctacg tcggcatgcg attgtttggt
     2461 ttagaagact gctttctaaa tatggttggg tgtatttaag ctagacccat acacccgctc
     2521 tatgggatta tttacttgtt tgaattttaa gatttgtgat aatggaactg gacgcaaaca
     2581 tttgatggaa aacgcatgtc atcattaacg aggtaacgta ggtatctgtc ctgccttagt
     2641 attgcacgca gcttcccagg acgcctagct attttttcat ctattcccct ctgtagtaac
     2701 gtaagagttt tcaagttttt aattcagact ttctcttcct ttgtttccaa tttccttcct
     2761 tactgcttga taccttttca atcccaaaga aaccgtgttc tttatatatt gtcgattgaa
     2821 agttacctac atcaactttc cgtgttccat tccgactata acaaacaacc aataagctca
     2881 actaattaag taaagcttat gtttcaacga aagactttac agagaaggaa cttgaaaggg
     2941 ctcaatctta acctgcaccc agatgtgggc aataatggcc aattgcagga aaagacatct
     3001 agacaacaaa tgggtcgtgg ttcggaattt gaagaactag gttccccgct aaaaaaacta
     3061 aaaatatcac cagatacggc ttctggtttg gttgaattcc aacaaatggg tcgtggttcg
     3121 gaatttgaag aactaggttc cccgctaaaa aaactaaaaa tatcaccaga tacggcttct
     3181 ggtttggtta gatctgcttc taaaggtgaa gaattattca ctggtgttgt cccaattttg
     3241 gttgaattag atggtgatgt taatggtcac aaattttctg tctccggtga aggtgaaggt
     3301 gatgctactt acggtaaatt gaccttaaaa tttatttgta ctactggtaa attgccagtt
     3361 ccatggccaa ccttagtcac tactttaggt tatggtttga tgtgttttgc tagataccca
     3421 gatcatatga aacaacatga ctttttcaag tctgccatgc cagaaggtta tgttcaagaa
     3481 agaactattt ttttcaaaga tgacggtaac tacaagacca gagctgaagt caagtttgaa
     3541 ggtgatacct tagttaatag aatcgaatta aaaggtattg attttaaaga agatggtaac
     3601 attttaggtc acaaattgga atacaactat aactctcaca atgtttacat catggctgac
     3661 aaacaaaaga atggtatcaa agttaacttc aaaattagac acaacattga agatggttct
     3721 gttcaattag ctgaccatta tcaacaaaat actccaattg gtgatggtcc agtcttgtta
     3781 ccagacaacc attacttatc ctatcaatct aaattatcca aagatccaaa cgaaaagaga
     3841 gaccacatgg tcttgaaaga atttgttact gctgctggta ttacccatgg tatggatgaa
     3901 ttgtacaaag gatcctagct cgagtcatgt aattagttat gtcacgctta cattcacgcc
     3961 ctccccccac atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc
     4021 ctatttattt ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc
     4081 ttttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga
     4141 aggttttggg acgctcgaag gctttaattt gcggccggta cccagctttt gttcccttta
     4201 gtgagggtta attccgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4261 ttatccgctc acaattccac acaacatagg agccggaagc ataaagtgta aagcctgggg
     4321 tgcctaatga gtgaggtaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4381 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4441 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4501 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4561 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4621 cgcgttgctg gcgtttttcc ataggctcgg cccccctgac gagcatcaca aaaatcgacg
     4681 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt tcccccctgg
     4741 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4801 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt
     4861 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4921 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4981 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5041 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     5101 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5161 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5221 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5281 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5341 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5401 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5461 ctgactgccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5521 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5581 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5641 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5701 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5761 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgaaaaa aagcggttag
     5821 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5881 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5941 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6001 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6061 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6121 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6181 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6241 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6301 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6361 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     6421 ctataaaaat aggcgtatca cgaggccctt tcgtc
//
