LOCUS       Exported                6558 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  SKARS: Fus3 MAPK activity sensor: Far1DS 2xNLS mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    pSP303 pRS306 pRPS2-Far1DS-NLS-mCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6558)
  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 6558)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6558
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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        265..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             3381..4082
                     /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(3520..3538)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3788..3807
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      4098..4345
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4114..4132)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(4368..4386)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4407..4423)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4407..4423)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4420..4442)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4431..4447
                     /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(4455..4485)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4638..4655)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4809..5397)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5568..6428)
                     /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     6191..6210
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6429..6533)
                     /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 gcttatgttt caacgaaaga ctttacagaa aagaggcaat attcctaaac
     3121 cactgaatct ttccctgcac ccagatgtgg gcaataatgg ccaattgcag gaaaagacat
     3181 ctagacaaca aatgggtcgt ggttcggaat ttgaagaact aggttccccg ctaaaaaaac
     3241 taaaaatatc accagatacg gcttctggtt tggttgaatt ccaacaaatg ggtcgtggtt
     3301 cggaatttga agaactaggt tccccgctaa aaaaactaaa aatatcacca gatacggctt
     3361 ctggtttggt tagatctgct agcaagggcg aggaggataa catggccatc atcaaggagt
     3421 tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg
     3481 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     3541 gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg
     3601 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct
     3661 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact
     3721 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct
     3781 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt
     3841 accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg
     3901 gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg
     3961 gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg
     4021 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     4081 agggatctta actcgagtca tgtaattagt tatgtcacgc ttacattcac gccctccccc
     4141 cacatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta
     4201 tttttttata gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt
     4261 tctgtacaga cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt
     4321 gggacgctcg aaggctttaa tttgcggccg gtacccagct tttgttccct ttagtgaggg
     4381 ttaattccga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4441 ctcacaattc cacacaacat aggagccgga agcataaagt gtaaagcctg gggtgcctaa
     4501 tgagtgaggt aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4561 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4621 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4681 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4741 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4801 ctggcgtttt tccataggct cggcccccct gacgagcatc acaaaaatcg acgctcaagt
     4861 cagaggtggc gaaacccgac aggactataa agataccagg cgttcccccc tggaagctcc
     4921 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4981 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     5041 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5101 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5161 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5221 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     5281 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5341 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5401 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5461 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5521 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5581 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactg
     5641 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5701 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5761 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5821 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5881 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5941 caacgatcaa ggcgagttac atgatccccc atgttgtgaa aaaaagcggt tagctccttc
     6001 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6061 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6121 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6181 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6241 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6301 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6361 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6421 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6481 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6541 ccccgaaaag tgccacct
//
