LOCUS       sequence-222024-        5164 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a regulatable secretory cargo (lacking an ER export 
            signal) for yeast at the TRP1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5164)
  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 5164)
  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..5164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(34..56)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     156..175
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        299..729
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             806..1477
                     /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(983..1003)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1201..1220
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1395..1418
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1490..1810
                     /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      1824..2071
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1840..1858)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2090..2106)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2090..2106)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2103..2125)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2114..2130
                     /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(2138..2144,2145..2162,2163..2168))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2183..2204
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2321..2338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2492..3080)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2572..2591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3251..4042,4043..4111))
                     /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     3874..3893
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4112..4216)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4284..4302
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4304..4404
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             4405..5079
                     /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 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
       61 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
      121 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
      181 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
      241 gcggacctta atacattcag acacttctgc ggtatcaccc tacttattcc cttcgagatt
      301 atatctagga acccatcagg ttggtggaag attacccgtt ctaagacttt tcagcttcct
      361 ctattgatgt tacacctgga cacccctttt ctggcatcca gtttttaatc ttcagtggca
      421 tgtgagattc tccgaaatta attaaagcaa ccacacaatt ctctcggata ccacctcggt
      481 tgaaactgac aggtggtttg ttacgcatgc taatgcaaag gagcctatat acctttggct
      541 cggctgctgt aacagggaat ataaagggca gcataattta ggagtttagt gaacttgcaa
      601 catttactat tttcccttct tacgtaaata tttttctttt taattctaaa tcaatctttt
      661 tcaatttttt gtttgtattc ttttcttgct taaatctata actacaaaaa acacatacat
      721 aaactaaaag aattcaaaaa tgaggcaggt ttggttctct tggattgtgg gattgttcct
      781 atgttttttc aacgtgtctt ctgctgatag cactgagaac gtcatcaagc ccttcatgcg
      841 cttcaaggtg cacatggagg gctcagtaaa tggtcatgag ttcgagatcg agggcgaggg
      901 cgagggcaag ccctacgagg gcacccagac cgccaagctg caggtgacca agggcggccc
      961 cctgcccttc gcctgggaca tcctgtcccc ccagttccag tacggctcca aggtgtacgt
     1021 gaagcacccc gccgacatcc ccgactacaa gaagctgtcc ttccccgagg gcttcaagtg
     1081 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
     1141 gcaggacggc accttcatct accacgtgaa gttcatcggc gtgaacttcc cctccgacgg
     1201 ccccgtaatg cagaagaaga ctctgggctg ggagccctcc accgagcgcc tgtacccccg
     1261 cgacggcgtg ctgaagggcg agatccacaa ggcgctgaag ctgaagggcg gcggccacta
     1321 cctggtggag ttcaagtcaa tctacatggc caagaagccc gtgaagctgc ccggctacta
     1381 ctacgtggac tccaagctgg acatcacctc ccacaacgag gactacaccg tggtggagca
     1441 gtacgagcgc gccgaggccc gccaccacct gttccaggga tcctctagag gagtgcaggt
     1501 ggaaaccatc tccccaggag acgggcgcac cttccccaag cgcggccaga ccgttgtggt
     1561 gcactacacc gggatgcttg aagatggaaa gaaactagat tcctcccggg acagaaacaa
     1621 gccctttaag tttatgctag gcaagcagga ggtgatccga ggctgggaag aaggggttgc
     1681 ccagatgagt gtgggtcaga gagccaaact gactatatct ccagattatg cctatggtgc
     1741 cactgggcac ccaggcgtta tcccaccaca tgccactctc gtcttcgatg tggagcttct
     1801 aaaactggaa taaactagag tcatcatgta attagttatg tcacgcttac attcacgccc
     1861 tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc
     1921 tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct
     1981 tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa
     2041 ggttttggga cgctcgaagg ctttaatttg caagcttggc gtaatcatgg tcatagctgt
     2101 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     2161 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     2221 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     2281 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2341 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2401 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2461 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2521 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2581 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2641 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2701 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2761 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2821 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2881 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2941 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3001 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3061 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3121 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3181 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3241 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3301 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3361 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3421 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3481 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3541 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3601 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3661 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3721 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3781 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3841 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3901 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3961 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4021 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4081 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4141 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4201 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     4261 ttatcatgac attaacctat aaaaataggc gtatcacgag gccaattcgg tcgaaaaaga
     4321 aaaggagagg gccaagaggg agggcattgg tgactattga gcacgtgagt atacgtgatt
     4381 aagcacacaa aggcagcttg gagtatgtct gttattaatt tcacaggtag ttctggtcca
     4441 ttggtgaaag tttgcggctt gcagagcaca gaggccgcag aatgtgcact agattccgat
     4501 gctgacttgc tgggtattat atgtgtgccc aatagaaaga gaacaattga cccggttatt
     4561 gcaaggaaaa tttcaagtct tgtaaaagca tataaaaata gttcaggcac tccgaaatac
     4621 ttggttggcg tgtttcgtaa tcaacctaag gaggatgttt tggctctggt caatgattac
     4681 ggcattgata tcgtccaact gcatggagat gagtcgtggc aagaatacca agagttcctc
     4741 ggtttgccag ttattaaaag actcgtattt ccaaaagact gcaacatact actcagtgca
     4801 gcttcacaga aacctcattc gtttattccc ttgtttgatt cagaagcagg tgggacaggt
     4861 gaacttttgg attggaactc gatttctgac tgggttggaa ggcaagagag ccccgagagc
     4921 ttacatttta tgttagctgg tggactgacg ccagaaaatg ttggtgatgc gcttagatta
     4981 aatggcgtta ttggtgttga tgtaagcgga ggtgtggaga caaatggtgt aaaagactct
     5041 aacaaaatag caaatttcgt caaaaatgct aagaaatagg ttattactga gtagtattta
     5101 tttaagtatt gtttgtgcac ttgcctgcaa gccttttgaa aagcaagcat aaaagatcgc
     5161 cctt
//
