LOCUS       Exported                8749 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  stable GFP reporter to monitor calcineurin activity via 
            calcineurin-dependent response element (CDRE) in S. cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    pAMS366-4xCDRE-GFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8749)
  AUTHORS   Diessl , J.,  Nandy, A., Schug, C., Habernig, L.,, Buttner, S.
  TITLE     Stable and destabilized GFP reporters to monitor calcineurin 
            activity in Saccharomyces cerevisiae.
  JOURNAL   Microbial Cell, 2020: in press
REFERENCE   2  (bases 1 to 8749)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8749
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        91..306
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             307..1110
                     /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"
     CDS             1582..2298
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(4315..4337)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4423..4563
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4478..4497
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4578..4595)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4738..5326)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4818..4837)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5497..6357)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6120..6139
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6358..6462)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6530..6548
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      6906..8248
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    7238..7285
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
        1 cggaagtgta tcgtacagta gacggagtat actagtatag tctatagtcc gtggaattct
       61 catgtttgac agcttatcat cgataagctt ttcaattcat catttttttt ttattctttt
      121 ttttgatttc ggtttccttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa
      181 cgaaggaagg agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca
      241 tgaaattgcc cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga
      301 taaatcatgt cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct
      361 gccaagctat ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt
      421 cgtaccacca aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta
      481 aaaacacatg tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag
      541 gcattatccg ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt
      601 aatacagtca aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt
      661 acgaatgcac acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa
      721 gaagtaacaa aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc
      781 ctatctactg gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt
      841 gttatcggct ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg
      901 attatgacac ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga
      961 accgtggatg atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt
     1021 gcaaagggaa gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca
     1081 tatttgagaa gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata
     1141 ctaaactcac aaattagagc ttcaatttaa ttatatcagt tattaccctc gagcaagcgc
     1201 acagccaccg actggtgtcg agcaagcgca cagccaccga ctggtgtcga ggtcgagcaa
     1261 gcgcacagcc accgactggt gtcgaggtcg agcaagcgca cagccaccga ctggtgtcga
     1321 ggtcgagcag atccgccagg cgtgtatata gcgtggatgg ccaggcaact ttagtgctga
     1381 cacatacagg catatatata tgtgtgcgac gacacatgat catatggcat gcatgtgctc
     1441 tgtatgtata taaaactctt gttttcttct tttctctaaa tattctttcc ttatacatta
     1501 ggtcctttgt agcataaatt actatacttc tatagacacg caaacacaaa tacacacact
     1561 aaattaataa tgaccggatc catgtctaaa ggtgaagaat tattcactgg tgttgtccca
     1621 attttggttg aattagatgg tgatgttaat ggtcacaaat tttctgtctc cggtgaaggt
     1681 gaaggtgatg ctacttacgg taaattgacc ttaaaattta tttgtactac tggtaaattg
     1741 ccagttccat ggccaacctt agtcactact ttcggttatg gtgttcaatg ttttgcgaga
     1801 tacccagatc atatgaaaca acatgacttt ttcaagtctg ccatgccaga aggttatgtt
     1861 caagaaagaa ctattttttt caaagatgac ggtaactaca agaccagagc tgaagtcaag
     1921 tttgaaggtg ataccttagt taatagaatc gaattaaaag gtattgattt taaagaagat
     1981 ggtaacattt taggtcacaa attggaatac aactataact ctcacaatgt ttacatcatg
     2041 gctgacaaac aaaagaatgg tatcaaagtt aacttcaaaa ttagacacaa cattgaagat
     2101 ggttctgttc aattagctga ccattatcaa caaaatactc caattggtga tggtccagtc
     2161 ttgttaccag acaaccatta cttatccact caatctgcct tatccaaaga tccaaacgaa
     2221 aagagagacc acatggtctt gttagaattt gttactgctg ctggtattac ccatggtatg
     2281 gatgaattgt acaaataaga gctcctgcac tggatggtgg cgctggatgg taagccgctg
     2341 gcaagcggtg aagtgcctct ggatgtcgct ccacaaggta aacagttgat tgaactgcct
     2401 gaactaccgc agccggagag cgccgggcaa ctctggctca cagtacgcgt agtgcaaccg
     2461 aacgcgaccg catggtcaga agccgggcac atcagcgcct ggcagcagtg gcgtctggcg
     2521 gaaaacctca gtgtgacgct ccccgccgcg tcccacgcca tcccgcatct gaccaccagc
     2581 gaaatggatt tttgcatcga gctgggtaat aagcgttggc aatttaaccg ccagtcaggc
     2641 tttctttcac agatgtggat tggcgataaa aaacaactgc tgacgccgct gcgcgatcag
     2701 ttcacccgtg caccgctgga taacgacatt ggcgtaagtg aagcgacccg cattgaccct
     2761 aacgcctggg tcgaacgctg gaaggcggcg ggccattacc aggccgaagc agcgttgttg
     2821 cagtgcacgg cagatacact tgctgatgcg gtgctgatta cgaccgctca cgcgtggcag
     2881 catcagggga aaaccttatt tatcagccgg aaaacctacc ggattgatgg tagtggtcaa
     2941 atggcgatta ccgttgatgt tgaagtggcg agcgatacac cgcatccggc gcggattggc
     3001 ctgaactgcc agctggcgca ggtagcagag cgggtaaact ggctcggatt agggccgcaa
     3061 gaaaactatc ccgaccgcct tactgccgcc tgttttgacc gctgggatct gccattgtca
     3121 gacatgtata ccccgtacgt cttcccgagc gaaaacggtc tgcgctgcgg gacgcgcgaa
     3181 ttgaattatg gcccacacca gtggcgcggc gacttccagt tcaacatcag ccgctacagt
     3241 caacagcaac tgatggaaac cagccatcgc catctgctgc acgcggaaga aggcacatgg
     3301 ctgaatatcg acggtttcca tatggggatt ggtggcgacg actcctggag cccgtcagta
     3361 tcggcggaat tccagctgag cgccggtcgc taccattacc agttggtctg gtgtcaaaaa
     3421 taataataac cgggcaggcc atgtctgccc gtatttcgcg taaggaaatc cattgtactg
     3481 ccggaccacc gactgtgagc cactccggcc atggcgtacg cactgacctg cttactgatt
     3541 tgtaaaaccg gtccggccat cacgctcaca taacgtccac gcaggctctc atagtgaaac
     3601 gtatcctccc cggtcatcac tgtgctgctc tttttcgacg cggcgaaccc cagggaagcc
     3661 atcaccccca cactgtccgt cagctcataa cggtacttca cgttaatccc tttcagatga
     3721 ctcacaccgg tatccccgcc cgacaacgac ggcaatgtac ccggtttcac ttgaaaatag
     3781 cccaccgtaa acgtaccatg tccaccttcc gcacgggccg gagtgactgt caccgcaagt
     3841 gcggcaaaga cagcaacggc aatacacaca ttacgcatcg ttcacctctc actgttttat
     3901 aataaaacgc ccgttcccgg acgaacctct gtaacacact cagaccacgc tgatgcccag
     3961 cgcctgtttc ttaatcacca taacctgcac atcgctggca aacgtatacg gcggaatatc
     4021 tgccgaatgc cgtgtggacg taagcgtgaa cgtcaggatc acgtttcccc gacccgctgg
     4081 catgtcaaca atacgggaga acacctgtac cgcctcgttc gccgcgccat cataaatcac
     4141 cgcaccgttc atcagtactt tcagataaca catcgaatac gttgtcctgc cgctgacagt
     4201 acgcttactt ccgcgaaacg tcagcggaag caccactatc tggcgatcaa aaggatggtc
     4261 atcggtcacg gtgacagtac gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     4321 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     4381 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     4441 acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
     4501 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     4561 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4621 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4681 ggaaagaaca tgtgagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4741 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4801 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4861 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4921 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4981 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5041 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5101 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5161 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5221 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5281 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5341 cttttctacg gtgtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5401 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5461 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5521 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5581 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5641 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5701 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5761 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat
     5821 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5881 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5941 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6001 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6061 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga
     6121 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6181 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6241 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6301 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6361 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6421 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6481 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     6541 cacgaggccc tttcgtcttc aagaattctg aaccagtcct aaaacgagta aataggaccg
     6601 gcaattcttc aagcaataaa caggaatacc aattattaaa agataactta gtcagatcgt
     6661 acaataaagc tttgaagaaa aatgcgcctt attcaatctt tgctataaaa aatggcccaa
     6721 aatctcacat tggaagacat ttgatgacct catttctttc aatgaagggc ctaacggagt
     6781 tgactaatgt tgtgggaaat tggagcgata agcgtgcttc tgccgtggcc aggacaacgt
     6841 atactcatca gataacagca atacctgatc actacttcgc actagtttct cggtactatg
     6901 catatgatcc aatatcaaag gaaatgatag cattgaagga tgagactaat ccaattgagg
     6961 agtggcagca tatagaacag ctaaagggta gtgctgaagg aagcatacga taccccgcat
     7021 ggaatgggat aatatcacag gaggtactag actacctttc atcctacata aatagacgca
     7081 tataagtacg catttaagca taaacacgca ctatgccgtt cttctcatgt atatatatat
     7141 acaggcaaca cgcagatata ggtgcgacgt gaacagtgag ctgtatgtgc gcagctcgcg
     7201 ttgcattttc ggaagcgctc gttttcggaa acgctttgaa gttcctattc cgaagttcct
     7261 attctctaga aagtatagga acttcagagc gcttttgaaa accaaaagcg ctctgaagac
     7321 gcactttcaa aaaaccaaaa acgcaccgga ctgtaacgag ctactaaaat attgcgaata
     7381 ccgcttccac aaacattgct caaaagtatc tctttgctat atatctctgt gctatatccc
     7441 tatataacct acccatccac ctttcgctcc ttgaacttgc atctaaactc gacctctaca
     7501 ttttttatgt ttatctctag tattactctt tagacaaaaa aattgtagta agaactattc
     7561 atagagtgaa tcgaaaacaa tacgaaaatg taaacatttc ctatacgtag tatatagaga
     7621 caaaatagaa gaaaccgttc ataattttct gaccaatgaa gaatcatcaa cgctatcact
     7681 ttctgttcac aaagtatgcg caatccacat cggtatagaa tataatcggg gatgccttta
     7741 tcttgaaaaa atgcacccgc agcttcgcta gtaatcagta aacgcgggaa gtggagtcag
     7801 gcttttttta tggaagagaa aatagacacc aaagtagcct tcttctaacc ttaacggacc
     7861 tacagtgcaa aaagttatca agagactgca ttatagagcg cacaaaggag aaaaaaagta
     7921 atctaagatg ctttgttaga aaaatagcgc tctcgggatg catttttgta gaacaaaaaa
     7981 gaagtataga ttctttgttg gtaaaatagc gctctcgcgt tgcatttctg ttctgtaaaa
     8041 atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt ttgttttaca
     8101 aaaatgaagc acagattctt cgttggtaaa atagcgcttt cgcgttgcat ttctgttctg
     8161 taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg catttttgtt
     8221 ctacaaaatg aagcacagat gcttcgttaa caaagatatg ctattgaagt gcaagatgga
     8281 aacgcagaaa atgaaccggg gatgcgacgt gcaagattac ctatgcaata gatgcaatag
     8341 tttctccagg aaccgaaata catacattgt cttccgtaaa gcgctagact atatattatt
     8401 atacaggttc aaatatacta tctgtttcag ggaaaactcc caggttcgga tgttcaaaat
     8461 tcaatgatgg gtaacaagta cgatcgtaaa tctgtaaaac agtttgtcgg atattaggct
     8521 gtatctcctc aaagcgtatt cgaatatcat tgagaagctg cagcgtcaca tcggataata
     8581 atgatggcag ccattgtaga agtgcctttt gcatttctag tctctttctc ggtctagcta
     8641 gttttactac atcgcgaaga tagaatctta gatcacactg cctttgctga gctggatcaa
     8701 tagagtaaca aaagagtggt aaggcctcgt taaaggacaa ggacctgag
//
