LOCUS       sequence-275883-        5929 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 5929)
  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 5929)
  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..5929
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1216..1238)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1338..1357
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1481..1911
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             2006..2677
                     /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     2401..2420
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2595..2618
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             2690..3010
                     /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      3024..3271
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3040..3058)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3290..3306)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3290..3306)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3303..3325)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3314..3330
                     /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(3338..3344,3345..3362,3363..3368))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3383..3404
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3521..3538)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3692..4280)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3772..3791)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4451..5242,5243..5311))
                     /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     5074..5093
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5312..5416)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5484..5502
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5504..5909
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             join(5910..5929,1..1075)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
ORIGIN
        1 cgtcgttttg ccaggtgacc acgttggtca agaaatcaca gccgaagcca ttaaggttct
       61 taaagctatt tctgatgttc gttccaatgt caagttcgat ttcgaaaatc atttaattgg
      121 tggtgctgct atcgatgcta caggtgttcc acttccagat gaggcgctgg aagcctccaa
      181 gaaggctgat gccgttttgt taggtgctgt gggtggtcct aaatggggta caggtagtgt
      241 tagacctgaa caaggtttac taaaaatccg taaagaactt caattgtacg ccaacttaag
      301 accatgtaac tttgcatccg actctctttt agacttatct ccaatcaagc cacaatttgc
      361 taaaggtact gacttcgttg ttgtcagaga attagtggga ggtatttact ttggtaagag
      421 aaaggaagac gatggtgatg gtgtcgcttg ggatagtgaa caatacaccg ttccagaagt
      481 gcaaagaatc acaagaatgg ccgctttcat ggccctacaa catgagccac cattgcctat
      541 ttggtccttg gataaagcta atgttttggc ctcttcaaga ttatggagaa aaactgtgga
      601 ggaaaccatc aagaacgaat ttcctacatt gaaggttcaa catcaattga ttgattctgc
      661 cgccatgatc ctagttaaga acccaaccca cctaaatggt attataatca ccagcaacat
      721 gtttggtgat atcatctccg atgaagcctc cgttatccca ggttccttgg gtttgttgcc
      781 atctgcgtcc ttggcctctt tgccagacaa gaacaccgca tttggtttgt acgaaccatg
      841 ccacggttct gctccagatt tgccaaagaa taaggtcaac cctatcgcca ctatcttgtc
      901 tgctgcaatg atgttgaaat tgtcattgaa cttgcctgaa gaaggtaagg ccattgaaga
      961 tgcagttaaa aaggttttgg atgcaggtat cagaactggt gatttaggtg gttccaacag
     1021 taccaccgaa gtcggtgatg ctgtcgccga agaagttaag aaaatccttg cttaaaaaga
     1081 ttctcttttt ttatgatatt tgtacataaa ctttataaat gaaattcata atagaaacga
     1141 cacgaaatta caaaatggaa tatgttcata gggtaggccc tttcgtctcg cgcgtttcgg
     1201 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     1261 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     1321 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg
     1381 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcggacct taatacattc
     1441 agacacttct gcggtatcac cctacttatt cccttcgaga ttatatctag gaacccatca
     1501 ggttggtgga agattacccg ttctaagact tttcagcttc ctctattgat gttacacctg
     1561 gacacccctt ttctggcatc cagtttttaa tcttcagtgg catgtgagat tctccgaaat
     1621 taattaaagc aatcacacaa ttctctcgga taccacctcg gttgaaactg acaggtggtt
     1681 tgttacgcat gctaatgcaa aggagcctat atacctttgg ctcggctgct gtaacaggga
     1741 atataaaggg cagcataatt taggagttta gtgaacttgc aacatttact attttccctt
     1801 cttacgtaaa tatttttctt tttaattcta aatcaatctt tttcaatttt ttgtttgtat
     1861 tcttttcttg cttaaatcta taactacaaa aaacacatac ataaactaaa agaattcaaa
     1921 aatgaggcag gtttggttct cttggattgt gggattgttc ctatgttttt tcaacgtgtc
     1981 ttctgctgcg ccggtgaaca ccaccgatag cactgagaac gtcatcaagc ccttcatgcg
     2041 cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg
     2101 cgagggcaag ccctacgagg gcacccagac cgccaagctg caggtgacca agggcggccc
     2161 cctgcccttc gcctgggaca tcctgtcccc ccagttcacc tacggctcca aggcctacgt
     2221 gaagcacccc gccgacatcc ccgactacaa gaagctgtcc ttccccgagg gcttcaagtg
     2281 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
     2341 gcaggacggc accttcatct accacgtgaa gttcatcggc gtgaacttcc cctccgacgg
     2401 ccccgtaatg cagaagaaga ctctgggctg ggagccctcc accgagcgcc tgtacccccg
     2461 cgacggcgtg ctgaagggcg agatccacaa ggcgctgaag ctgaagggcg gcggccacta
     2521 cctggtggag ttcaagacaa tctacatggc caagaagccc gtgaagctgc ccggctacta
     2581 ctacgtggac tccaagctgg acatcacctc ccacaacgag gactacaccg tggtggagca
     2641 gtacgagcgc gccgaggccc gccaccacct gttccaggga tcctctagag gagtgcaggt
     2701 ggaaaccatc tccccaggag acgggcgcac cttccccaag cgcggccaga ccgttgtggt
     2761 gcactacacc gggatgcttg aagatggaaa gaaactagat tcctcccggg acagaaacaa
     2821 gccctttaag tttatgctag gcaagcagga ggtgatccga ggctgggaag aaggggttgc
     2881 ccagatgagt gtgggtcaga gagccaaact gactatatct ccagattatg cctatggtgc
     2941 cactgggcac ccaggcgtta tcccaccaca tgccactctc gtcttcgatg tggagcttct
     3001 aaaactggaa taaactagag tcatcatgta attagttatg tcacgcttac attcacgccc
     3061 tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc
     3121 tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct
     3181 tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa
     3241 ggttttggga cgctcgaagg ctttaatttg caagcttggc gtaatcatgg tcatagctgt
     3301 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     3361 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     3421 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     3481 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     3541 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3601 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3661 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3721 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3781 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3841 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3901 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3961 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4021 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4081 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4141 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4201 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4261 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4321 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4381 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4441 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4501 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4561 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4621 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4681 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4741 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4801 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4861 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4921 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4981 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5041 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5101 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5161 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5221 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5281 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5341 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5401 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     5461 ttatcatgac attaacctat aaaaataggc gtatcacgag gccaactgtg ggaatactca
     5521 ggtatcgtaa gatgcaagag ttcgaatctc ttagcaacca ttattttttt cctcaacata
     5581 acgagaacac acaggggcgc tatcgcacag aatcaaattc gatgactgga aattttttgt
     5641 taatttcaga ggtcgcctga cgcatatacc tttttcaact gaaaaattgg gagaaaaagg
     5701 aaaggtgaga gcgccggaac cggcttttca tatagaatag agaagcgttc atgactaaat
     5761 gcttgcatca caatacttga agttgacaat attatttaag gacctattgt tttttccaat
     5821 aggtggttag caatcgtctt actttctaac ttttcttacc ttttacattt cagcaatata
     5881 tatatatatt tcaaggatat accattctaa tgtctgcccc taagaagat
//
