LOCUS       sequence-222027-        5182 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a non-glycosylated 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-APVNTA-DsRed-Express2-FKBP(LV)...
FEATURES             Location/Qualifiers
     source          1..5182
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(30..52)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     152..171
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        295..725
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             820..1491
                     /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(997..1017)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1215..1234
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1409..1432
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1504..1824
                     /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      1838..2085
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1854..1872)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2104..2120)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2104..2120)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2117..2139)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2128..2144
                     /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(2152..2158,2159..2176,2177..2182))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2197..2218
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2335..2352)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2506..3094)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2586..2605)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3265..4056,4057..4125))
                     /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     3888..3907
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4126..4230)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4298..4316
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4318..4418
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             4419..5093
                     /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 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
       61 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
      121 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
      181 caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgg
      241 accttaatac attcagacac ttctgcggta tcaccctact tattcccttc gagattatat
      301 ctaggaaccc atcaggttgg tggaagatta cccgttctaa gacttttcag cttcctctat
      361 tgatgttaca cctggacacc ccttttctgg catccagttt ttaatcttca gtggcatgtg
      421 agattctccg aaattaatta aagcaaccac acaattctct cggataccac ctcggttgaa
      481 actgacaggt ggtttgttac gcatgctaat gcaaaggagc ctatatacct ttggctcggc
      541 tgctgtaaca gggaatataa agggcagcat aatttaggag tttagtgaac ttgcaacatt
      601 tactattttc ccttcttacg taaatatttt tctttttaat tctaaatcaa tctttttcaa
      661 ttttttgttt gtattctttt cttgcttaaa tctataacta caaaaaacac atacataaac
      721 taaaagaatt caaaaatgag gcaggtttgg ttctcttgga ttgtgggatt gttcctatgt
      781 tttttcaacg tgtcttctgc tgcgccggtg aacaccgcgg atagcactga gaacgtcatc
      841 aagcccttca tgcgcttcaa ggtgcacatg gagggctcag taaatggtca tgagttcgag
      901 atcgagggcg agggcgaggg caagccctac gagggcaccc agaccgccaa gctgcaggtg
      961 accaagggcg gccccctgcc cttcgcctgg gacatcctgt ccccccagtt ccagtacggc
     1021 tccaaggtgt acgtgaagca ccccgccgac atccccgact acaagaagct gtccttcccc
     1081 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     1141 caggactcct ccctgcagga cggcaccttc atctaccacg tgaagttcat cggcgtgaac
     1201 ttcccctccg acggccccgt aatgcagaag aagactctgg gctgggagcc ctccaccgag
     1261 cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc acaaggcgct gaagctgaag
     1321 ggcggcggcc actacctggt ggagttcaag tcaatctaca tggccaagaa gcccgtgaag
     1381 ctgcccggct actactacgt ggactccaag ctggacatca cctcccacaa cgaggactac
     1441 accgtggtgg agcagtacga gcgcgccgag gcccgccacc acctgttcca gggatcctct
     1501 agaggagtgc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc
     1561 cagaccgttg tggtgcacta caccgggatg cttgaagatg gaaagaaact agattcctcc
     1621 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg
     1681 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat
     1741 tatgcctatg gtgccactgg gcacccaggc gttatcccac cacatgccac tctcgtcttc
     1801 gatgtggagc ttctaaaact ggaataaact agagtcatca tgtaattagt tatgtcacgc
     1861 ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct
     1921 gaagtctagg tccctattta tttttttata gttatgttag tattaagaac gttatttata
     1981 tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa
     2041 accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcaagct tggcgtaatc
     2101 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     2161 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     2221 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     2281 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     2341 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     2401 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2461 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2521 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2581 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2641 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     2701 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     2761 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2821 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     2881 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     2941 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3001 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3061 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3121 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3181 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3241 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3301 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3361 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     3421 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3481 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     3541 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     3601 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     3661 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     3721 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     3781 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     3841 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     3901 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     3961 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4021 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4081 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4141 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4201 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     4261 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccaat
     4321 tcggtcgaaa aagaaaagga gagggccaag agggagggca ttggtgacta ttgagcacgt
     4381 gagtatacgt gattaagcac acaaaggcag cttggagtat gtctgttatt aatttcacag
     4441 gtagttctgg tccattggtg aaagtttgcg gcttgcagag cacagaggcc gcagaatgtg
     4501 cactagattc cgatgctgac ttgctgggta ttatatgtgt gcccaataga aagagaacaa
     4561 ttgacccggt tattgcaagg aaaatttcaa gtcttgtaaa agcatataaa aatagttcag
     4621 gcactccgaa atacttggtt ggcgtgtttc gtaatcaacc taaggaggat gttttggctc
     4681 tggtcaatga ttacggcatt gatatcgtcc aactgcatgg agatgagtcg tggcaagaat
     4741 accaagagtt cctcggtttg ccagttatta aaagactcgt atttccaaaa gactgcaaca
     4801 tactactcag tgcagcttca cagaaacctc attcgtttat tcccttgttt gattcagaag
     4861 caggtgggac aggtgaactt ttggattgga actcgatttc tgactgggtt ggaaggcaag
     4921 agagccccga gagcttacat tttatgttag ctggtggact gacgccagaa aatgttggtg
     4981 atgcgcttag attaaatggc gttattggtg ttgatgtaag cggaggtgtg gagacaaatg
     5041 gtgtaaaaga ctctaacaaa atagcaaatt tcgtcaaaaa tgctaagaaa taggttatta
     5101 ctgagtagta tttatttaag tattgtttgt gcacttgcct gcaagccttt tgaaaagcaa
     5161 gcataaaaga tcgccctttc gt
//
