LOCUS       sequence-275867-        5209 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a regulatable vacuolar cargo for yeast at the TRP1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5209)
  AUTHORS   Casler JC, Glick BS
  TITLE     A microscopy-based kinetic analysis of yeast vacuolar protein 
            sorting.
  JOURNAL   Elife. 2020 Jun 25;9. pii: 56844. doi: 10.7554/eLife.56844.
  PUBMED    32584255
REFERENCE   2  (bases 1 to 5209)
  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: "Elife."; 
            date: "2020-06-25"; volume: "9. pii: 56844. doi"; pages: " 
            10.7554/eLife.56844"
COMMENT     Sequence Label: 
            YIplac204-pOst1-APVNTT-DsRed-Express2-FKBP(LV)C22V-QRPL
FEATURES             Location/Qualifiers
     source          1..5209
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     34..52
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        54..154
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             155..829
                     /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"
     primer_bind     complement(948..970)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1070..1089
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1213..1643
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             1738..2409
                     /codon_start=1
                     /product="noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2008)"
                     /label=DsRed-Express2
                     /note="mammalian codon-optimized"
                     /translation="DSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAKL
                     QVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHKA
                     LKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHHL
                     FQ"
     primer_bind     complement(1915..1935)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2133..2152
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2327..2350
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             2422..2742
                     /codon_start=1
                     /product="destabilization domain that can be stabilized by 
                     Shield1 in the ProteoTuner(TM) system"
                     /label=DD
                     /note="L106P mutant of FKBP12"
                     /translation="GVQVETISPGDGRTFPKRGQTVVVHYTGMLEDGKKLDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGVIPPHATLVFDVELL
                     KLE"
     CDS             2422..2742
                     /codon_start=1
                     /product="dimeric F36M mutant of FK506-binding protein 
                     FKBP12"
                     /label=FKBP-M
                     /note="dimerization can be reversed by ligand binding"
                     /translation="GVQVETISPGDGRTFPKRGQTVVVHYTGMLEDGKKLDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGVIPPHATLVFDVELL
                     KLE"
     CDS             2422..2742
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTVVVHYTGMLEDGKKLDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGVIPPHATLVFDVELL
                     KLE"
     CDS             2422..2742
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTVVVHYTGMLEDGKKLDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGVIPPHATLVFDVELL
                     KLE"
     terminator      2783..3030
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2799..2817)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3049..3065)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3049..3065)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3062..3084)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3073..3089
                     /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(3097..3103,3104..3121,3122..3127))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3142..3163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3280..3297)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3451..4039)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3531..3550)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4210..5001,5002..5070))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4833..4852
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5071..5175)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccaattcgg
       61 tcgaaaaaga aaaggagagg gccaagaggg agggcattgg tgactattga gcacgtgagt
      121 atacgtgatt aagcacacaa aggcagcttg gagtatgtct gttattaatt tcacaggtag
      181 ttctggtcca ttggtgaaag tttgcggctt gcagagcaca gaggccgcag aatgtgcact
      241 agattccgat gctgacttgc tgggtattat atgtgtgccc aatagaaaga gaacaattga
      301 cccggttatt gcaaggaaaa tttcaagtct tgtaaaagca tataaaaata gttcaggcac
      361 tccgaaatac ttggttggcg tgtttcgtaa tcaacctaag gaggatgttt tggctctggt
      421 caatgattac ggcattgata tcgtccaact gcatggagat gagtcgtggc aagaatacca
      481 agagttcctc ggtttgccag ttattaaaag actcgtattt ccaaaagact gcaacatact
      541 actcagtgca gcttcacaga aacctcattc gtttattccc ttgtttgatt cagaagcagg
      601 tgggacaggt gaacttttgg attggaactc gatttctgac tgggttggaa ggcaagagag
      661 ccccgagagc ttacatttta tgttagctgg tggactgacg ccagaaaatg ttggtgatgc
      721 gcttagatta aatggcgtta ttggtgttga tgtaagcgga ggtgtggaga caaatggtgt
      781 aaaagactct aacaaaatag caaatttcgt caaaaatgct aagaaatagg ttattactga
      841 gtagtattta tttaagtatt gtttgtgcac ttgcctgcaa gccttttgaa aagcaagcat
      901 aaaagatcgc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
      961 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     1021 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     1081 cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     1141 aaataccgca tcaggcggac cttaatacat tcagacactt ctgcggtatc accctactta
     1201 ttcccttcga gattatatct aggaacccat caggttggtg gaagattacc cgttctaaga
     1261 cttttcagct tcctctattg atgttacacc tggacacccc ttttctggca tccagttttt
     1321 aatcttcagt ggcatgtgag attctccgaa attaattaaa gcaaccacac aattctctcg
     1381 gataccacct cggttgaaac tgacaggtgg tttgttacgc atgctaatgc aaaggagcct
     1441 atataccttt ggctcggctg ctgtaacagg gaatataaag ggcagcataa tttaggagtt
     1501 tagtgaactt gcaacattta ctattttccc ttcttacgta aatatttttc tttttaattc
     1561 taaatcaatc tttttcaatt ttttgtttgt attcttttct tgcttaaatc tataactaca
     1621 aaaaacacat acataaacta aaagaattca aaaatgaggc aggtttggtt ctcttggatt
     1681 gtgggattgt tcctatgttt tttcaacgtg tcttctgctg cgccggtgaa caccaccgat
     1741 agcactgaga acgtcatcaa gcccttcatg cgcttcaagg tgcacatgga gggctcagta
     1801 aatggtcatg agttcgagat cgagggcgag ggcgagggca agccctacga gggcacccag
     1861 accgccaagc tgcaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc
     1921 ccccagttcc agtacggctc caaggtgtac gtgaagcacc ccgccgacat ccccgactac
     1981 aagaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     2041 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcaccttcat ctaccacgtg
     2101 aagttcatcg gcgtgaactt cccctccgac ggccccgtaa tgcagaagaa gactctgggc
     2161 tgggagccct ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg cgagatccac
     2221 aaggcgctga agctgaaggg cggcggccac tacctggtgg agttcaagtc aatctacatg
     2281 gccaagaagc ccgtgaagct gcccggctac tactacgtgg actccaagct ggacatcacc
     2341 tcccacaacg aggactacac cgtggtggag cagtacgagc gcgccgaggc ccgccaccac
     2401 ctgttccagg gatcctctag aggagtgcag gtggaaacca tctccccagg agacgggcgc
     2461 accttcccca agcgcggcca gaccgttgtg gtgcactaca ccgggatgct tgaagatgga
     2521 aagaaactag attcctcccg ggacagaaac aagcccttta agtttatgct aggcaagcag
     2581 gaggtgatcc gaggctggga agaaggggtt gcccagatga gtgtgggtca gagagccaaa
     2641 ctgactatat ctccagatta tgcctatggt gccactgggc acccaggcgt tatcccacca
     2701 catgccactc tcgtcttcga tgtggagctt ctaaaactgg aaggatctca gagaccactg
     2761 ggaggatcct aaactagagt catcatgtaa ttagttatgt cacgcttaca ttcacgccct
     2821 ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct
     2881 atttattttt ttatagttat gttagtatta agaacgttat ttatatttca aatttttctt
     2941 ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag
     3001 gttttgggac gctcgaaggc tttaatttgc aagcttggcg taatcatggt catagctgtt
     3061 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     3121 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     3181 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3241 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     3301 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3361 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3421 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3481 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3541 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3601 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3661 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3721 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3781 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3841 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3901 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3961 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4021 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4081 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4141 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4201 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4261 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     4321 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     4381 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     4441 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     4501 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4561 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4621 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4681 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4741 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4801 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4861 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4921 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4981 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     5041 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     5101 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5161 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaa
//
