LOCUS       sequence-119441-        6753 bp    DNA     circular SYN 15-APR-2025
DEFINITION  SKARS: Mpk1 MAPK activity sensor: Mkk2DS (1-100) 2xNLS mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6753)
  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 6753)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pSP323 pRS306 pRPS2-Mkk2DS-NLS-mCherry
FEATURES             Location/Qualifiers
     source          1..6753
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     43..61
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(99..121)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     221..240
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        260..480
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             481..1284
                     /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(1413..1868)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1550..1571)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1762..1781
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1998..2020
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2012..2029
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2013..2029
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2036..2055
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2036..2054
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3576..4277
                     /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(3715..3733)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3983..4002
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      4293..4540
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4309..4327)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(4563..4581)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4602..4618)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4602..4618)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4615..4637)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4626..4642
                     /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(4650..4656,4657..4674,4675..4680))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4833..4850)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5004..5592)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5763..6554,6555..6623))
                     /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     6386..6405
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6624..6728)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
       61 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
      121 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
      181 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
      241 ctgagagtgc accacgcttt tcaattcaat tcatcatttt ttttttattc ttttttttga
      301 tttcggtttc tttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg
      361 aaggagcaca gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat
      421 tgcccagtat tcttaaccca actgcacaga acaaaaacct gcaggaaacg aagataaatc
      481 atgtcgaaag ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag
      541 ctatttaata tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc
      601 accaaggaat tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca
      661 catgtggata tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta
      721 tccgccaagt acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca
      781 gtcaaattgc agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat
      841 gcacacggtg tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta
      901 acaaaggaac ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct
      961 actggagaat atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc
     1021 ggctttattg ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg
     1081 acacccggtg tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg
     1141 gatgatgtgg tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag
     1201 ggaagggatg ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg
     1261 agaagatgcg gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac
     1321 tcacaaatta gagcttcaat ttaattatat cagttattac cctgcggtgt gaaataccgc
     1381 acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata ttttgttaaa
     1441 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     1501 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     1561 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     1621 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     1681 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     1741 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     1801 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     1861 gggcgcgtcg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     1921 ggcctcttcg ctattacgcc agctggcgaa ggggggatgt gctgcaaggc gattaagttg
     1981 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata
     2041 cgactcacta tagggcgaat tggagctcgt tgccacgatt ggaataccct caaaattccc
     2101 acttttacta tgcttcaatt gacccttcgg tatctcagca tctaggtagc tttcattgag
     2161 attcatagtt ccgatgtact gattctacgt ttgtgtactt tgatagactt ttttattcaa
     2221 ttcgtattca ctactatctt tgtctcgtac cgcggttttt atcctttatt tcagccctac
     2281 gagatagaac gatggagcct agtaatacca ttttatgtaa gcaaaataag aagaaagact
     2341 aaaagaatta ggatatgttg ttaatcaggg ataaaggtac gcatactagc cttactacgt
     2401 aaatataagg ttacggttta cgatcactat tgcaagatac tattgtttag cattggcaaa
     2461 gacttgacgg gaaatctttg attgggccca accatattca ttaaagatcc gggatgtgag
     2521 cttgaactgc ttttcgacgc gaagaaggtt tacacgtatt ttagtaattg gcttattcac
     2581 taaggattct taaggttttc ttaatagttt tctacgtcgg catgcgattg tttggtttag
     2641 aagactgctt tctaaatatg gttgggtgta tttaagctag acccatacac ccgctctatg
     2701 ggattattta cttgtttgaa ttttaagatt tgtgataatg gaactggacg caaacatttg
     2761 atggaaaacg catgtcatca ttaacgaggt aacgtaggta tctgtcctgc cttagtattg
     2821 cacgcagctt cccaggacgc ctagctattt tttcatctat tcccctctgt agtaacgtaa
     2881 gagttttcaa gtttttaatt cagactttct cttcctttgt ttccaatttc cttccttact
     2941 gcttgatacc ttttcaatcc caaagaaacc gtgttcttta tatattgtcg attgaaagtt
     3001 acctacatca actttccgtg ttccattccg actataacaa acaaccaata agctcaacta
     3061 attaagtaaa gcttatggct tcaatgttca gaccaccaga atccaatagg agtcaccaaa
     3121 agactccaaa attaacgctt ccagtaaatt tagttcaaaa tgcgaaatcc actaatgatg
     3181 ggcaacatct caaccggtca ccgtactcgt cagtgaacga aagcccatac tccaacaata
     3241 gcacttcagc tacttccact acgtcatcca tggcttcaaa ttccactttg ttgtacaata
     3301 gatcatctac tacaactatt aaaaatagac cggtaccacc tccattacct cccctagtac
     3361 taacgcaaaa aaaatctaga caacaaatgg gtcgtggttc ggaatttgaa gaactaggtt
     3421 ccccgctaaa aaaactaaaa atatcaccag atacggcttc tggtttggtt gaattccaac
     3481 aaatgggtcg tggttcggaa tttgaagaac taggttcccc gctaaaaaaa ctaaaaatat
     3541 caccagatac ggcttctggt ttggttagat ctgctagcaa gggcgaggag gataacatgg
     3601 ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg
     3661 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     3721 tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca
     3781 tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt
     3841 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
     3901 ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg
     3961 gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct
     4021 cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga
     4081 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc
     4141 ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg
     4201 aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca
     4261 tggacgagct gtacaaggga tcttaactcg agtcatgtaa ttagttatgt cacgcttaca
     4321 ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt
     4381 ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat ttatatttca
     4441 aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt
     4501 gcttgagaag gttttgggac gctcgaaggc tttaatttgc ggccggtacc cagcttttgt
     4561 tccctttagt gagggttaat tccgagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4621 tgaaattgtt atccgctcac aattccacac aacataggag ccggaagcat aaagtgtaaa
     4681 gcctggggtg cctaatgagt gaggtaactc acattaattg cgttgcgctc actgcccgct
     4741 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4801 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4861 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4921 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4981 aaaaaggccg cgttgctggc gtttttccat aggctcggcc cccctgacga gcatcacaaa
     5041 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttc
     5101 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5161 tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc
     5221 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5281 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5341 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5401 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     5461 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5521 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5581 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5641 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5701 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5761 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5821 atagttgcct gactgcccgt cgtgtagata actacgatac gggagggctt accatctggc
     5881 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5941 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6001 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6061 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6121 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgaaaaaaa
     6181 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6241 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6301 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6361 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6421 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6481 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6541 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6601 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6661 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6721 gttccgcgca catttccccg aaaagtgcca cct
//
