LOCUS       sequence-275871-        5209 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a 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-ESCargo(Crimson)
FEATURES             Location/Qualifiers
     source          1..5209
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..439
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             534..1205
                     /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(711..731)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     929..948
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1123..1146
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1218..1538
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTVVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGVIPPHATLVFDVELL
                     KLE"
     terminator      1579..1826
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1595..1613)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1845..1861)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1845..1861)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1858..1880)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1869..1885
                     /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(1893..1899,1900..1917,1918..1923))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1938..1959
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2076..2093)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2247..2835)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2327..2346)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3006..3797,3798..3866))
                     /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     3629..3648
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3867..3971)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4039..4057
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4059..4159
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             4160..4834
                     /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(4953..4975)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5075..5094
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cttcgagatt atatctagga acccatcagg ttggtggaag attacccgtt ctaagacttt
       61 tcagcttcct ctattgatgt tacacctgga cacccctttt ctggcatcca gtttttaatc
      121 ttcagtggca tgtgagattc tccgaaatta attaaagcaa ccacacaatt ctctcggata
      181 ccacctcggt tgaaactgac aggtggtttg ttacgcatgc taatgcaaag gagcctatat
      241 acctttggct cggctgctgt aacagggaat ataaagggca gcataattta ggagtttagt
      301 gaacttgcaa catttactat tttcccttct tacgtaaata tttttctttt taattctaaa
      361 tcaatctttt tcaatttttt gtttgtattc ttttcttgct taaatctata actacaaaaa
      421 acacatacat aaactaaaag aattcaaaaa tgaggcaggt ttggttctct tggattgtgg
      481 gattgttcct atgttttttc aacgtgtctt ctgctgcgcc ggtgaacacc accgatagca
      541 ctgagaacgt catcaagccc ttcatgcgct tcaaggtgca catggagggc tcagtaaatg
      601 gtcatgagtt cgagatcgag ggcgagggcg agggcaagcc ctacgagggc acccagaccg
      661 ccaagctgca ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc
      721 agttccagta cggctccaag gtgtacgtga agcaccccgc cgacatcccc gactacaaga
      781 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg
      841 tggtgaccgt gacccaggac tcctccctgc aggacggcac cttcatctac cacgtgaagt
      901 tcatcggcgt gaacttcccc tccgacggcc ccgtaatgca gaagaagact ctgggctggg
      961 agccctccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag atccacaagg
     1021 cgctgaagct gaagggcggc ggccactacc tggtggagtt caagtcaatc tacatggcca
     1081 agaagcccgt gaagctgccc ggctactact acgtggactc caagctggac atcacctccc
     1141 acaacgagga ctacaccgtg gtggagcagt acgagcgcgc cgaggcccgc caccacctgt
     1201 tccagggatc ctctagagga gtgcaggtgg aaaccatctc cccaggagac gggcgcacct
     1261 tccccaagcg cggccagacc gttgtggtgc actacaccgg gatgcttgaa gatggaaaga
     1321 aatttgattc ctcccgggac agaaacaagc cctttaagtt tatgctaggc aagcaggagg
     1381 tgatccgagg ctgggaagaa ggggttgccc agatgagtgt gggtcagaga gccaaactga
     1441 ctatatctcc agattatgcc tatggtgcca ctgggcaccc aggcgttatc ccaccacatg
     1501 ccactctcgt cttcgatgtg gagcttctaa aactggaagg atctcagaga ccactgggag
     1561 gatcctaaac tagagtcatc atgtaattag ttatgtcacg cttacattca cgccctcccc
     1621 ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt
     1681 atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt
     1741 ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt
     1801 tgggacgctc gaaggcttta atttgcaagc ttggcgtaat catggtcata gctgtttcct
     1861 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     1921 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     1981 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     2041 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     2101 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2161 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2221 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2281 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     2341 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2401 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     2461 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2521 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2581 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2641 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     2701 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2761 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2821 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     2881 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     2941 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     3001 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3061 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     3121 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     3181 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3241 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     3301 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     3361 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3421 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     3481 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     3541 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3601 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     3661 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3721 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     3781 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     3841 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     3901 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     3961 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     4021 atgacattaa cctataaaaa taggcgtatc acgaggccaa ttcggtcgaa aaagaaaagg
     4081 agagggccaa gagggagggc attggtgact attgagcacg tgagtatacg tgattaagca
     4141 cacaaaggca gcttggagta tgtctgttat taatttcaca ggtagttctg gtccattggt
     4201 gaaagtttgc ggcttgcaga gcacagaggc cgcagaatgt gcactagatt ccgatgctga
     4261 cttgctgggt attatatgtg tgcccaatag aaagagaaca attgacccgg ttattgcaag
     4321 gaaaatttca agtcttgtaa aagcatataa aaatagttca ggcactccga aatacttggt
     4381 tggcgtgttt cgtaatcaac ctaaggagga tgttttggct ctggtcaatg attacggcat
     4441 tgatatcgtc caactgcatg gagatgagtc gtggcaagaa taccaagagt tcctcggttt
     4501 gccagttatt aaaagactcg tatttccaaa agactgcaac atactactca gtgcagcttc
     4561 acagaaacct cattcgttta ttcccttgtt tgattcagaa gcaggtggga caggtgaact
     4621 tttggattgg aactcgattt ctgactgggt tggaaggcaa gagagccccg agagcttaca
     4681 ttttatgtta gctggtggac tgacgccaga aaatgttggt gatgcgctta gattaaatgg
     4741 cgttattggt gttgatgtaa gcggaggtgt ggagacaaat ggtgtaaaag actctaacaa
     4801 aatagcaaat ttcgtcaaaa atgctaagaa ataggttatt actgagtagt atttatttaa
     4861 gtattgtttg tgcacttgcc tgcaagcctt ttgaaaagca agcataaaag atcgcccttt
     4921 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     4981 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     5041 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     5101 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     5161 cggaccttaa tacattcaga cacttctgcg gtatcaccct acttattcc
//
