LOCUS       sequence-221987-        5182 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 5182)
  AUTHORS   Casler JC, Papanikou E, Barrero JJ, Glick BS
  TITLE     Maturation-driven transport and AP-1-dependent recycling of a 
            secretory cargo in the Golgi.
  JOURNAL   J Cell Biol. 2019 May 6;218(5):1582-1601. doi: 
            10.1083/jcb.201807195. Epub 2019 Mar 11.
  PUBMED    30858194
REFERENCE   2  (bases 1 to 5182)
  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"; doi: 
            "10.1083/jcb.201807195"; journalName: "J Cell Biol"; date: 
            "2019-05-6- 6"; volume: "218"; issue: "5"; pages: "1582-1601"
COMMENT     Sequence Label: YIplac204-pOst1-ESCargo
FEATURES             Location/Qualifiers
     source          1..5182
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(38..60)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     160..179
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        303..733
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             828..1499
                     /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(1005..1025)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1223..1242
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1417..1440
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1512..1832
                     /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      1846..2093
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1862..1880)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2112..2128)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2112..2128)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2125..2147)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2136..2152
                     /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(2160..2166,2167..2184,2185..2190))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2205..2226
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2343..2360)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2514..3102)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2594..2613)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3273..4064,4065..4133))
                     /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     3896..3915
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4134..4238)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4306..4324
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4326..4426
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             4427..5101
                     /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 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
       61 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
      121 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
      181 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
      241 tcaggcggac cttaatacat tcagacactt ctgcggtatc accctactta ttcccttcga
      301 gattatatct aggaacccat caggttggtg gaagattacc cgttctaaga cttttcagct
      361 tcctctattg atgttacacc tggacacccc ttttctggca tccagttttt aatcttcagt
      421 ggcatgtgag attctccgaa attaattaaa gcaaccacac aattctctcg gataccacct
      481 cggttgaaac tgacaggtgg tttgttacgc atgctaatgc aaaggagcct atataccttt
      541 ggctcggctg ctgtaacagg gaatataaag ggcagcataa tttaggagtt tagtgaactt
      601 gcaacattta ctattttccc ttcttacgta aatatttttc tttttaattc taaatcaatc
      661 tttttcaatt ttttgtttgt attcttttct tgcttaaatc tataactaca aaaaacacat
      721 acataaacta aaagaattca aaaatgaggc aggtttggtt ctcttggatt gtgggattgt
      781 tcctatgttt tttcaacgtg tcttctgctg cgccggtgaa caccaccgat agcactgaga
      841 acgtcatcaa gcccttcatg cgcttcaagg tgcacatgga gggctcagta aatggtcatg
      901 agttcgagat cgagggcgag ggcgagggca agccctacga gggcacccag accgccaagc
      961 tgcaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc ccccagttcc
     1021 agtacggctc caaggtgtac gtgaagcacc ccgccgacat ccccgactac aagaagctgt
     1081 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
     1141 ccgtgaccca ggactcctcc ctgcaggacg gcaccttcat ctaccacgtg aagttcatcg
     1201 gcgtgaactt cccctccgac ggccccgtaa tgcagaagaa gactctgggc tgggagccct
     1261 ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg cgagatccac aaggcgctga
     1321 agctgaaggg cggcggccac tacctggtgg agttcaagtc aatctacatg gccaagaagc
     1381 ccgtgaagct gcccggctac tactacgtgg actccaagct ggacatcacc tcccacaacg
     1441 aggactacac cgtggtggag cagtacgagc gcgccgaggc ccgccaccac ctgttccagg
     1501 gatcctctag aggagtgcag gtggaaacca tctccccagg agacgggcgc accttcccca
     1561 agcgcggcca gaccgttgtg gtgcactaca ccgggatgct tgaagatgga aagaaactag
     1621 attcctcccg ggacagaaac aagcccttta agtttatgct aggcaagcag gaggtgatcc
     1681 gaggctggga agaaggggtt gcccagatga gtgtgggtca gagagccaaa ctgactatat
     1741 ctccagatta tgcctatggt gccactgggc acccaggcgt tatcccacca catgccactc
     1801 tcgtcttcga tgtggagctt ctaaaactgg aataaactag agtcatcatg taattagtta
     1861 tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta
     1921 gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt
     1981 tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta
     2041 tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcaagcttg
     2101 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     2161 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     2221 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2281 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2341 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2401 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2461 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2521 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2581 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2641 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2701 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2761 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2821 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2881 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2941 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3001 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3061 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3121 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3181 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3241 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3301 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3361 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     3421 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3481 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3541 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3601 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3661 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3721 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3781 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3841 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3901 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3961 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4021 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4081 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4141 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4201 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4261 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     4321 aggccaattc ggtcgaaaaa gaaaaggaga gggccaagag ggagggcatt ggtgactatt
     4381 gagcacgtga gtatacgtga ttaagcacac aaaggcagct tggagtatgt ctgttattaa
     4441 tttcacaggt agttctggtc cattggtgaa agtttgcggc ttgcagagca cagaggccgc
     4501 agaatgtgca ctagattccg atgctgactt gctgggtatt atatgtgtgc ccaatagaaa
     4561 gagaacaatt gacccggtta ttgcaaggaa aatttcaagt cttgtaaaag catataaaaa
     4621 tagttcaggc actccgaaat acttggttgg cgtgtttcgt aatcaaccta aggaggatgt
     4681 tttggctctg gtcaatgatt acggcattga tatcgtccaa ctgcatggag atgagtcgtg
     4741 gcaagaatac caagagttcc tcggtttgcc agttattaaa agactcgtat ttccaaaaga
     4801 ctgcaacata ctactcagtg cagcttcaca gaaacctcat tcgtttattc ccttgtttga
     4861 ttcagaagca ggtgggacag gtgaactttt ggattggaac tcgatttctg actgggttgg
     4921 aaggcaagag agccccgaga gcttacattt tatgttagct ggtggactga cgccagaaaa
     4981 tgttggtgat gcgcttagat taaatggcgt tattggtgtt gatgtaagcg gaggtgtgga
     5041 gacaaatggt gtaaaagact ctaacaaaat agcaaatttc gtcaaaaatg ctaagaaata
     5101 ggttattact gagtagtatt tatttaagta ttgtttgtgc acttgcctgc aagccttttg
     5161 aaaagcaagc ataaaagatc gc
//
