LOCUS       sequence-271645-        5927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a regulatable secretory cargo for yeast at the LEU2 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5927)
  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 5927)
  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: YIplac128-pOst1-ESCargo(Orange)
FEATURES             Location/Qualifiers
     source          1..5927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1016..1038)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1138..1157
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1281..1711
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             1806..2477
                     /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     2201..2220
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2395..2418
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             2490..2810
                     /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      2824..3071
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2840..2858)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3090..3106)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3090..3106)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3103..3125)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3114..3130
                     /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(3138..3144,3145..3162,3163..3168))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3183..3204
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3320..3337)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3491..4079)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3571..3590)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4250..5041,5042..5110))
                     /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     4873..4892
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5111..5215)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5283..5301
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5303..5707
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             join(5708..5927,1..875)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
ORIGIN
        1 taggtgctgt gggtggtcct aaatggggta caggtagtgt tagacctgaa caaggtttac
       61 taaaaatccg taaagaactt caattgtacg ccaacttaag accatgtaac tttgcatccg
      121 actctctttt agacttatct ccaatcaagc cacaatttgc taaaggtact gacttcgttg
      181 ttgtcagaga attagtggga ggtatttact ttggtaagag aaaggaagac gatggtgatg
      241 gtgtcgcttg ggatagtgaa caatacaccg ttccagaagt gcaaagaatc acaagaatgg
      301 ccgctttcat ggccctacaa catgagccac cattgcctat ttggtccttg gataaagcta
      361 atgttttggc ctcttcaaga ttatggagaa aaactgtgga ggaaaccatc aagaacgaat
      421 ttcctacatt gaaggttcaa catcaattga ttgattctgc cgccatgatc ctagttaaga
      481 acccaaccca cctaaatggt attataatca ccagcaacat gtttggtgat atcatctccg
      541 atgaagcctc cgttatccca ggttccttgg gtttgttgcc atctgcgtcc ttggcctctt
      601 tgccagacaa gaacaccgca tttggtttgt acgaaccatg ccacggttct gctccagatt
      661 tgccaaagaa taaggttgac cctatcgcca ctatcttgtc tgctgcaatg atgttgaaat
      721 tgtcattgaa cttgcctgaa gaaggtaagg ccattgaaga tgcagttaaa aaggttttgg
      781 atgcaggtat cagaactggt gatttaggtg gttccaacag taccaccgaa gtcggtgatg
      841 ctgtcgccga agaagttaag aaaatccttg cttaaaaaga ttctcttttt ttatgatatt
      901 tgtacataaa ctttataaat gaaattcata atagaaacga cacgaaatta caaaatggaa
      961 tatgttcata gggtaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     1021 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     1081 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     1141 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
     1201 taaggagaaa ataccgcatc aggcggacct taatacattc agacacttct gcggtatcac
     1261 cctacttatt cccttcgaga ttatatctag gaacccatca ggttggtgga agattacccg
     1321 ttctaagact tttcagcttc ctctattgat gttacacctg gacacccctt ttctggcatc
     1381 cagtttttaa tcttcagtgg catgtgagat tctccgaaat taattaaagc aatcacacaa
     1441 ttctctcgga taccacctcg gttgaaactg acaggtggtt tgttacgcat gctaatgcaa
     1501 aggagcctat atacctttgg ctcggctgct gtaacaggga atataaaggg cagcataatt
     1561 taggagttta gtgaacttgc aacatttact attttccctt cttacgtaaa tatttttctt
     1621 tttaattcta aatcaatctt tttcaatttt ttgtttgtat tcttttcttg cttaaatcta
     1681 taactacaaa aaacacatac ataaactaaa agaattcaaa aatgaggcag gtttggttct
     1741 cttggattgt gggattgttc ctatgttttt tcaacgtgtc ttctgctgcg ccggtgaaca
     1801 ccaccgatag cactgagaac gtcatcaagc ccttcatgcg cttcaaggtg cacatggagg
     1861 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggcaag ccctacgagg
     1921 gcacccagac cgccaagctg caggtgacca agggcggccc cctgcccttc gcctgggaca
     1981 tcctgtcccc ccagttcacc tacggctcca aggcctacgt gaagcacccc gccgacatcc
     2041 ccgactacaa gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     2101 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc accttcatct
     2161 accacgtgaa gttcatcggc gtgaacttcc cctccgacgg ccccgtaatg cagaagaaga
     2221 ctctgggctg ggagccctcc accgagcgcc tgtacccccg cgacggcgtg ctgaagggcg
     2281 agatccacaa ggcgctgaag ctgaagggcg gcggccacta cctggtggag ttcaagacaa
     2341 tctacatggc caagaagccc gtgaagctgc ccggctacta ctacgtggac tccaagctgg
     2401 acatcacctc ccacaacgag gactacaccg tggtggagca gtacgagcgc gccgaggccc
     2461 gccaccacct gttccaggga tcctctagag gagtgcaggt ggaaaccatc tccccaggag
     2521 acgggcgcac cttccccaag cgcggccaga ccgttgtggt gcactacacc gggatgcttg
     2581 aagatggaaa gaaactagat tcctcccggg acagaaacaa gccctttaag tttatgctag
     2641 gcaagcagga ggtgatccga ggctgggaag aaggggttgc ccagatgagt gtgggtcaga
     2701 gagccaaact gactatatct ccagattatg cctatggtgc cactgggcac ccaggcgtta
     2761 tcccaccaca tgccactctc gtcttcgatg tggagcttct aaaactggaa taaactagag
     2821 tcatcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa
     2881 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
     2941 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
     3001 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg
     3061 ctttaatttg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3121 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     3181 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3241 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3301 attgggcgtc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3361 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3421 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3481 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3541 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3601 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3661 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3721 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3781 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3841 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     3901 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     3961 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4021 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4081 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4141 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4201 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4261 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4321 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4381 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4441 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4501 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4561 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4621 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4681 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4741 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4801 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     4861 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     4921 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4981 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5041 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5101 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5161 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5221 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     5281 aaaataggcg tatcacgagg ccaactgtgg gaatactcag gtatcgtaag atgcaagagt
     5341 tcgaatctct tagcaaccat tatttttttc ctcaacataa cgagaacaca caggggcgct
     5401 atcgcacaga atcaaattcg atgactggaa attttttgtt aatttcagag gtcgcctgac
     5461 gcatatacct ttttcaactg aaaaattggg agaaaaagga aaggtgagag gccggaaccg
     5521 gcttttcata tagaatagag aagcgttcat gactaaatgc ttgcatcaca atacttgaag
     5581 ttgacaatat tatttaagga cctattgttt tttccaatag gtggttagca atcgtcttac
     5641 tttctaactt ttcttacctt ttacatttca gcaatatata tatatatttc aaggatatac
     5701 cattctaatg tctgccccta agaagatcgt cgttttgcca ggtgaccacg ttggtcaaga
     5761 aatcacagcc gaagccatta aggttcttaa agctatttct gatgttcgtt ccaatgtcaa
     5821 gttcgatttc gaaaatcatt taattggtgg tgctgctatc gatgctacag gtgtcccact
     5881 tccagatgag gcgctggaag cctccaagaa ggttgatgcc gttttgt
//
