LOCUS       sequence-271638-        5210 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 5210)
  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 5210)
  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..5210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..29)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(183..771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(263..282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(942..1733,1734..1802))
                     /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     1565..1584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1803..1907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1975..1993
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1995..2096
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             2097..2771
                     /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(2890..2912)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3012..3031
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3155..3585
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             3680..4351
                     /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(3857..3877)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     4075..4094
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4269..4292
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             4364..4684
                     /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             4364..4684
                     /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             4364..4684
                     /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             4364..4684
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTVVVHYTGMLEDGKKLDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGVIPPHATLVFDVELL
                     KLE"
     terminator      4725..4972
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4741..4759)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4991..5007)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4991..5007)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5004..5026)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5015..5031
                     /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(5039..5045,5046..5063,5064..5069))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5084..5105
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
       61 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      181 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      601 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
      661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      721 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1081 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1801 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1861 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1921 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     1981 cgtatcacga ggccaattcg gtcgaaaaaa gaaaaggaga gggccaagag ggagggcatt
     2041 ggtgactatt gagcacgtga gtatacgtga ttaagcacac aaaggcagct tggagtatgt
     2101 ctgttattaa tttcacaggt agttctggtc cattggtgaa agtttgcggc ttgcagagca
     2161 cagaggccgc agaatgtgca ctagattccg atgctgactt gctgggtatt atatgtgtgc
     2221 ccaatagaaa gagaacaatt gacccggtta ttgcaaggaa aatttcaagt cttgtaaaag
     2281 catataaaaa tagttcaggc actccgaaat acttggttgg cgtgtttcgt aatcaaccta
     2341 aggaggatgt tttggctctg gtcaatgatt acggcattga tatcgtccaa ctgcatggag
     2401 atgagtcgtg gcaagaatac caagagttcc tcggtttgcc agttattaaa agactcgtat
     2461 ttccaaaaga ctgcaacata ctactcagtg cagcttcaca gaaacctcat tcgtttattc
     2521 ccttgtttga ttcagaagca ggtgggacag gtgaactttt ggattggaac tcgatttctg
     2581 actgggttgg aaggcaagag agccccgaga gcttacattt tatgttagct ggtggactga
     2641 cgccagaaaa tgttggtgat gcgcttagat taaatggcgt tattggtgtt gatgtaagcg
     2701 gaggtgtgga gacaaatggt gtaaaagact ctaacaaaat agcaaatttc gtcaaaaatg
     2761 ctaagaaata ggttattact gagtagtatt tatttaagta ttgtttgtgc acttgcctgc
     2821 aagccttttg aaaagcaagc ataaaagatc gccctttcgt ctcgcgcgtt tcggtgatga
     2881 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     2941 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
     3001 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
     3061 accgcacaga tgcgtaagga gaaaataccg catcaggcgg accttaatac attcagacac
     3121 ttctgcggta tcaccctact tattcccttc gagattatat ctaggaaccc atcaggttgg
     3181 tggaagatta cccgttctaa gacttttcag cttcctctat tgatgttaca cctggacacc
     3241 ccttttctgg catccagttt ttaatcttca gtggcatgtg agattctccg aaattaatta
     3301 aagcaatcac acaattctct cggataccac ctcggttgaa actgacaggt ggtttgttac
     3361 gcatgctaat gcaaaggagc ctatatacct ttggctcggc tgctgtaaca gggaatataa
     3421 agggcagcat aatttaggag tttagtgaac ttgcaacatt tactattttc ccttcttacg
     3481 taaatatttt tctttttaat tctaaatcaa tctttttcaa ttttttgttt gtattctttt
     3541 cttgcttaaa tctataacta caaaaaacac atacataaac taaaagaatt caaaaatgag
     3601 gcaggtttgg ttctcttgga ttgtgggatt gttcctatgt tttttcaacg tgtcttctgc
     3661 tgcgccggtg aacaccaccg atagcactga gaacgtcatc aagcccttca tgcgcttcaa
     3721 ggtgcacatg gagggctcag taaatggtca tgagttcgag atcgagggcg agggcgaggg
     3781 caagccctac gagggcaccc agaccgccaa gctgcaggtg accaagggcg gccccctgcc
     3841 cttcgcctgg gacatcctgt ccccccagtt ccagtacggc tccaaggtgt acgtgaagca
     3901 ccccgccgac atccccgact acaagaagct gtccttcccc gagggcttca agtgggagcg
     3961 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     4021 cggcaccttc atctaccacg tgaagttcat cggcgtgaac ttcccctccg acggccccgt
     4081 aatgcagaag aagactctgg gctgggagcc ctccaccgag cgcctgtacc cccgcgacgg
     4141 cgtgctgaag ggcgagatcc acaaggcgct gaagctgaag ggcggcggcc actacctggt
     4201 ggagttcaag tcaatctaca tggccaagaa gcccgtgaag ctgcccggct actactacgt
     4261 ggactccaag ctggacatca cctcccacaa cgaggactac accgtggtgg agcagtacga
     4321 gcgcgccgag gcccgccacc acctgttcca gggatcctct agaggagtgc aggtggaaac
     4381 catctcccca ggagacgggc gcaccttccc caagcgcggc cagaccgttg tggtgcacta
     4441 caccgggatg cttgaagatg gaaagaaact agattcctcc cgggacagaa acaagccctt
     4501 taagtttatg ctaggcaagc aggaggtgat ccgaggctgg gaagaagggg ttgcccagat
     4561 gagtgtgggt cagagagcca aactgactat atctccagat tatgcctatg gtgccactgg
     4621 gcacccaggc gttatcccac cacatgccac tctcgtcttc gatgtggagc ttctaaaact
     4681 ggaaggatct cagagaccac tgggaggatc ctaaactaga gtcatcatgt aattagttat
     4741 gtcacgctta cattcacgcc ctccccccac atccgctcta accgaaaagg aaggagttag
     4801 acaacctgaa gtctaggtcc ctatttattt ttttatagtt atgttagtat taagaacgtt
     4861 atttatattt caaatttttc ttttttttct gtacagacgc gtgtacgcat gtaacattat
     4921 actgaaaacc ttgcttgaga aggttttggg acgctcgaag gctttaattt gcaagcttgg
     4981 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5041 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5101 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5161 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
//
