LOCUS       sequence-275877-        5183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a regulatable secretory cargo for yeast at the TRP1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5183)
  AUTHORS   Casler JC, Zajac AL, Valbuena FM, Sparvoli D, Jeyifous O, Turkewitz 
            AP, Horne-Badovinac S, Green WN, Glick BS
  TITLE     ESCargo: a regulatable fluorescent secretory cargo for diverse model
            organisms.
  JOURNAL   Mol Biol Cell. 2020 Oct 28:mbcE20090591. doi: 
            10.1091/mbc.E20-09-0591.
  PUBMED    33112725
REFERENCE   2  (bases 1 to 5183)
  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: "Mol Biol 
            Cell. 2020 Oct 28:mbcE20090591. doi: 10.1091/mbc.E20-09-0591."
COMMENT     Sequence Label: YIplac204-pOst1-APVNTT-E2-Crimson-FKBP(LV)(C22V)
FEATURES             Location/Qualifiers
     source          1..5183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(52..74)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     174..193
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        317..747
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             842..1513
                     /codon_start=1
                     /product="far-red noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2009)"
                     /label=E2-Crimson
                     /note="mammalian codon-optimized"
                     /translation="DSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAKL
                     QVTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHMA
                     LKLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHHL
                     FQ"
     primer_bind     1237..1256
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1526..1846
                     /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"
     terminator      1860..2107
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1876..1894)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2126..2142)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2126..2142)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2139..2161)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2150..2166
                     /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(2174..2180,2181..2198,2199..2204))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2219..2240
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2357..2374)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2528..3116)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2608..2627)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3287..4078,4079..4147))
                     /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     3910..3929
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4148..4252)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4320..4338
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4340..4441
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             4442..5116
                     /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"
ORIGIN
        1 gcataaaaga tcgccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
       61 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
      121 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
      181 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
      241 gagaaaatac cgcatcaggc ggaccttaat acattcagac acttctgcgg tatcacccta
      301 cttattccct tcgagattat atctaggaac ccatcaggtt ggtggaagat tacccgttct
      361 aagacttttc agcttcctct attgatgtta cacctggaca ccccttttct ggcatccagt
      421 ttttaatctt cagtggcatg tgagattctc cgaaattaat taaagcaacc acacaattct
      481 ctcggatacc acctcggttg aaactgacag gtggtttgtt acgcatgcta atgcaaagga
      541 gcctatatac ctttggctcg gctgctgtaa cagggaatat aaagggcagc ataatttagg
      601 agtttagtga acttgcaaca tttactattt tcccttctta cgtaaatatt tttcttttta
      661 attctaaatc aatctttttc aattttttgt ttgtattctt ttcttgctta aatctataac
      721 tacaaaaaac acatacataa actaaaagaa ttcaaaaatg aggcaggttt ggttctcttg
      781 gattgtggga ttgttcctat gttttttcaa cgtgtcttct gctgcgccgg tgaacaccac
      841 cgatagcact gagaacgtca tcaagccctt catgcgcttc aaggtgcaca tggagggctc
      901 cgtgaacggc cacgagttcg agatcgaggg cgtgggcgag ggcaagccct acgagggcac
      961 ccagaccgcc aagctgcaag tgaccaaggg cggccccctg cccttcgcct gggacatcct
     1021 gtccccccag ttcttctacg gctccaaggc gtacatcaag caccccgccg acatccccga
     1081 ctacctcaag cagtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     1141 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcaccc tcatctacca
     1201 cgtgaagttc atcggcgtga acttcccctc cgacggcccc gtaatgcaga agaagactct
     1261 gggctgggag ccctccactg agcgcaacta cccccgcgac ggcgtgctga agggcgagaa
     1321 ccacatggcg ctgaagctga agggcggcgg ccactacctg tgtgagttca agtccatcta
     1381 catggccaag aagcccgtga agctgcccgg ctaccactac gtggactaca agctcgacat
     1441 cacctcccac aacgaggact acaccgtggt ggagcagtac gagcgcgccg aggcccgcca
     1501 ccacctgttc cagggatcct ctagaggagt gcaggtggaa accatctccc caggagacgg
     1561 gcgcaccttc cccaagcgcg gccagaccgt tgtggtgcac tacaccggga tgcttgaaga
     1621 tggaaagaaa ctagattcct cccgggacag aaacaagccc tttaagttta tgctaggcaa
     1681 gcaggaggtg atccgaggct gggaagaagg ggttgcccag atgagtgtgg gtcagagagc
     1741 caaactgact atatctccag attatgccta tggtgccact gggcacccag gcgttatccc
     1801 accacatgcc actctcgtct tcgatgtgga gcttctaaaa ctggaataaa ctagagtcat
     1861 catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg ctctaaccga
     1921 aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt
     1981 agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta
     2041 cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt
     2101 aatttgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     2161 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     2221 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     2281 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     2341 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     2401 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2461 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2521 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2581 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2641 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2701 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     2761 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2821 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2881 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2941 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     3001 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3061 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3121 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     3181 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     3241 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     3301 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     3361 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     3421 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3481 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3541 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3601 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3661 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3721 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3781 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3841 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3901 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3961 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4021 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4081 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4141 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     4201 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     4261 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     4321 ataggcgtat cacgaggcca attcggtcga aaaaagaaaa ggagagggcc aagagggagg
     4381 gcattggtga ctattgagca cgtgagtata cgtgattaag cacacaaagg cagcttggag
     4441 tatgtctgtt attaatttca caggtagttc tggtccattg gtgaaagttt gcggcttgca
     4501 gagcacagag gccgcagaat gtgcactaga ttccgatgct gacttgctgg gtattatatg
     4561 tgtgcccaat agaaagagaa caattgaccc ggttattgca aggaaaattt caagtcttgt
     4621 aaaagcatat aaaaatagtt caggcactcc gaaatacttg gttggcgtgt ttcgtaatca
     4681 acctaaggag gatgttttgg ctctggtcaa tgattacggc attgatatcg tccaactgca
     4741 tggagatgag tcgtggcaag aataccaaga gttcctcggt ttgccagtta ttaaaagact
     4801 cgtatttcca aaagactgca acatactact cagtgcagct tcacagaaac ctcattcgtt
     4861 tattcccttg tttgattcag aagcaggtgg gacaggtgaa cttttggatt ggaactcgat
     4921 ttctgactgg gttggaaggc aagagagccc cgagagctta cattttatgt tagctggtgg
     4981 actgacgcca gaaaatgttg gtgatgcgct tagattaaat ggcgttattg gtgttgatgt
     5041 aagcggaggt gtggagacaa atggtgtaaa agactctaac aaaatagcaa atttcgtcaa
     5101 aaatgctaag aaataggtta ttactgagta gtatttattt aagtattgtt tgtgcacttg
     5161 cctgcaagcc ttttgaaaag caa
//
