LOCUS       sequence-275930-        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-ESCargo(Orange)
FEATURES             Location/Qualifiers
     source          1..5183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        12..113
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             114..788
                     /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(907..929)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1029..1048
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1172..1602
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             1697..2368
                     /codon_start=1
                     /product="noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2008)"
                     /label=DsRed-Express2
                     /note="mammalian codon-optimized"
                     /translation="DSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAKL
                     QVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHKA
                     LKLKGGGHYLVEFKTIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHHL
                     FQ"
     primer_bind     2092..2111
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2286..2309
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             2381..2701
                     /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      2715..2962
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2731..2749)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2981..2997)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2981..2997)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2994..3016)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3005..3021
                     /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(3029..3035,3036..3053,3054..3059))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3074..3095
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3212..3229)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3383..3971)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3463..3482)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4142..4933,4934..5002))
                     /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     4765..4784
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5003..5107)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(5175..5183,1..10)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 atcacgaggc caattcggtc gaaaaaagaa aaggagaggg ccaagaggga gggcattggt
       61 gactattgag cacgtgagta tacgtgatta agcacacaaa ggcagcttgg agtatgtctg
      121 ttattaattt cacaggtagt tctggtccat tggtgaaagt ttgcggcttg cagagcacag
      181 aggccgcaga atgtgcacta gattccgatg ctgacttgct gggtattata tgtgtgccca
      241 atagaaagag aacaattgac ccggttattg caaggaaaat ttcaagtctt gtaaaagcat
      301 ataaaaatag ttcaggcact ccgaaatact tggttggcgt gtttcgtaat caacctaagg
      361 aggatgtttt ggctctggtc aatgattacg gcattgatat cgtccaactg catggagatg
      421 agtcgtggca agaataccaa gagttcctcg gtttgccagt tattaaaaga ctcgtatttc
      481 caaaagactg caacatacta ctcagtgcag cttcacagaa acctcattcg tttattccct
      541 tgtttgattc agaagcaggt gggacaggtg aacttttgga ttggaactcg atttctgact
      601 gggttggaag gcaagagagc cccgagagct tacattttat gttagctggt ggactgacgc
      661 cagaaaatgt tggtgatgcg cttagattaa atggcgttat tggtgttgat gtaagcggag
      721 gtgtggagac aaatggtgta aaagactcta acaaaatagc aaatttcgtc aaaaatgcta
      781 agaaataggt tattactgag tagtatttat ttaagtattg tttgtgcact tgcctgcaag
      841 ccttttgaaa agcaagcata aaagatcgcc ctttcgtctc gcgcgtttcg gtgatgacgg
      901 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
      961 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
     1021 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
     1081 gcacagatgc gtaaggagaa aataccgcat caggcggacc ttaatacatt cagacacttc
     1141 tgcggtatca ccctacttat tcccttcgag attatatcta ggaacccatc aggttggtgg
     1201 aagattaccc gttctaagac ttttcagctt cctctattga tgttacacct ggacacccct
     1261 tttctggcat ccagttttta atcttcagtg gcatgtgaga ttctccgaaa ttaattaaag
     1321 caaccacaca attctctcgg ataccacctc ggttgaaact gacaggtggt ttgttacgca
     1381 tgctaatgca aaggagccta tatacctttg gctcggctgc tgtaacaggg aatataaagg
     1441 gcagcataat ttaggagttt agtgaacttg caacatttac tattttccct tcttacgtaa
     1501 atatttttct ttttaattct aaatcaatct ttttcaattt tttgtttgta ttcttttctt
     1561 gcttaaatct ataactacaa aaaacacata cataaactaa aagaattcaa aaatgaggca
     1621 ggtttggttc tcttggattg tgggattgtt cctatgtttt ttcaacgtgt cttctgctgc
     1681 gccggtgaac accaccgata gcactgagaa cgtcatcaag cccttcatgc gcttcaaggt
     1741 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggcaa
     1801 gccctacgag ggcacccaga ccgccaagct gcaggtgacc aagggcggcc ccctgccctt
     1861 cgcctgggac atcctgtccc cccagttcac ctacggctcc aaggcctacg tgaagcaccc
     1921 cgccgacatc cccgactaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     1981 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     2041 caccttcatc taccacgtga agttcatcgg cgtgaacttc ccctccgacg gccccgtaat
     2101 gcagaagaag actctgggct gggagccctc caccgagcgc ctgtaccccc gcgacggcgt
     2161 gctgaagggc gagatccaca aggcgctgaa gctgaagggc ggcggccact acctggtgga
     2221 gttcaagaca atctacatgg ccaagaagcc cgtgaagctg cccggctact actacgtgga
     2281 ctccaagctg gacatcacct cccacaacga ggactacacc gtggtggagc agtacgagcg
     2341 cgccgaggcc cgccaccacc tgttccaggg atcctctaga ggagtgcagg tggaaaccat
     2401 ctccccagga gacgggcgca ccttccccaa gcgcggccag accgttgtgg tgcactacac
     2461 cgggatgctt gaagatggaa agaaactaga ttcctcccgg gacagaaaca agccctttaa
     2521 gtttatgcta ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag
     2581 tgtgggtcag agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca
     2641 cccaggcgtt atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga
     2701 ataaactaga gtcatcatgt aattagttat gtcacgctta cattcacgcc ctccccccac
     2761 atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt
     2821 ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct
     2881 gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg
     2941 acgctcgaag gctttaattt gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     3001 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     3061 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     3121 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3181 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3241 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3301 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3361 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3421 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3481 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3541 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3601 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3661 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3721 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3781 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     3841 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3901 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3961 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4021 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4081 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4141 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4201 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4261 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4321 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4381 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4441 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4501 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4561 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4621 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4681 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4741 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4801 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4861 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4921 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     4981 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5041 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5101 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     5161 cattaaccta taaaaatagg cgt
//
