LOCUS       sequence-275881-        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(Crimson)
FEATURES             Location/Qualifiers
     source          1..5929
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(146..168)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     268..287
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        411..841
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             936..1607
                     /codon_start=1
                     /product="far-red noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2009)"
                     /label=E2-Crimson
                     /note="mammalian codon-optimized"
                     /translation="DSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAKL
                     QVTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHMA
                     LKLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHHL
                     FQ"
     primer_bind     1331..1350
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1620..1940
                     /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      1954..2201
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1970..1988)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2220..2236)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2220..2236)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2233..2255)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2244..2260
                     /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(2268..2274,2275..2292,2293..2298))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2313..2334
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2451..2468)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2622..3210)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2702..2721)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3381..4172,4173..4241))
                     /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     4004..4023
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4242..4346)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4414..4432
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4434..4839
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             join(4840..5929,1..5)
                     /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 cttaaaaaga ttctcttttt ttatgatatt tgtacataaa ctttataaat gaaattcata
       61 atagaaacga cacgaaatta caaaatggaa tatgttcata gggtaggccc tttcgtctcg
      121 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
      181 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
      241 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
      301 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcggacct
      361 taatacattc agacacttct gcggtatcac cctacttatt cccttcgaga ttatatctag
      421 gaacccatca ggttggtgga agattacccg ttctaagact tttcagcttc ctctattgat
      481 gttacacctg gacacccctt ttctggcatc cagtttttaa tcttcagtgg catgtgagat
      541 tctccgaaat taattaaagc aatcacacaa ttctctcgga taccacctcg gttgaaactg
      601 acaggtggtt tgttacgcat gctaatgcaa aggagcctat atacctttgg ctcggctgct
      661 gtaacaggga atataaaggg cagcataatt taggagttta gtgaacttgc aacatttact
      721 attttccctt cttacgtaaa tatttttctt tttaattcta aatcaatctt tttcaatttt
      781 ttgtttgtat tcttttcttg cttaaatcta taactacaaa aaacacatac ataaactaaa
      841 agaattcaaa aatgaggcag gtttggttct cttggattgt gggattgttc ctatgttttt
      901 tcaacgtgtc ttctgctgcg ccggtgaaca ccaccgatag cactgagaac gtcatcaagc
      961 ccttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg
     1021 agggcgtggg cgagggcaag ccctacgagg gcacccagac cgccaagctg caagtgacca
     1081 agggcggccc cctgcccttc gcctgggaca tcctgtcccc ccagttcttc tacggctcca
     1141 aggcgtacat caagcacccc gccgacatcc ccgactacct caagcagtcc ttccccgagg
     1201 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
     1261 actcctccct gcaggacggc accctcatct accacgtgaa gttcatcggc gtgaacttcc
     1321 cctccgacgg ccccgtaatg cagaagaaga ctctgggctg ggagccctcc actgagcgca
     1381 actacccccg cgacggcgtg ctgaagggcg agaaccacat ggcgctgaag ctgaagggcg
     1441 gcggccacta cctgtgtgag ttcaagtcca tctacatggc caagaagccc gtgaagctgc
     1501 ccggctacca ctacgtggac tacaagctcg acatcacctc ccacaacgag gactacaccg
     1561 tggtggagca gtacgagcgc gccgaggccc gccaccacct gttccaggga tcctctagag
     1621 gagtgcaggt ggaaaccatc tccccaggag acgggcgcac cttccccaag cgcggccaga
     1681 ccgttgtggt gcactacacc gggatgcttg aagatggaaa gaaactagat tcctcccggg
     1741 acagaaacaa gccctttaag tttatgctag gcaagcagga ggtgatccga ggctgggaag
     1801 aaggggttgc ccagatgagt gtgggtcaga gagccaaact gactatatct ccagattatg
     1861 cctatggtgc cactgggcac ccaggcgtta tcccaccaca tgccactctc gtcttcgatg
     1921 tggagcttct aaaactggaa taaactagag tcatcatgta attagttatg tcacgcttac
     1981 attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag
     2041 tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc
     2101 aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct
     2161 tgcttgagaa ggttttggga cgctcgaagg ctttaatttg caagcttggc gtaatcatgg
     2221 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     2281 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     2341 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     2401 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     2461 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     2521 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     2581 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     2641 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     2701 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     2761 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     2821 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     2881 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     2941 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3001 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3061 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3121 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3181 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3241 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3301 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3361 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3421 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     3481 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     3541 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3601 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3661 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3721 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     3781 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     3841 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     3901 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     3961 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     4021 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     4081 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4141 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4201 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4261 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4321 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     4381 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccaactgtg
     4441 ggaatactca ggtatcgtaa gatgcaagag ttcgaatctc ttagcaacca ttattttttt
     4501 cctcaacata acgagaacac acaggggcgc tatcgcacag aatcaaattc gatgactgga
     4561 aattttttgt taatttcaga ggtcgcctga cgcatatacc tttttcaact gaaaaattgg
     4621 gagaaaaagg aaaggtgaga gcgccggaac cggcttttca tatagaatag agaagcgttc
     4681 atgactaaat gcttgcatca caatacttga agttgacaat attatttaag gacctattgt
     4741 tttttccaat aggtggttag caatcgtctt actttctaac ttttcttacc ttttacattt
     4801 cagcaatata tatatatatt tcaaggatat accattctaa tgtctgcccc taagaagatc
     4861 gtcgttttgc caggtgacca cgttggtcaa gaaatcacag ccgaagccat taaggttctt
     4921 aaagctattt ctgatgttcg ttccaatgtc aagttcgatt tcgaaaatca tttaattggt
     4981 ggtgctgcta tcgatgctac aggtgttcca cttccagatg aggcgctgga agcctccaag
     5041 aaggctgatg ccgttttgtt aggtgctgtg ggtggtccta aatggggtac aggtagtgtt
     5101 agacctgaac aaggtttact aaaaatccgt aaagaacttc aattgtacgc caacttaaga
     5161 ccatgtaact ttgcatccga ctctctttta gacttatctc caatcaagcc acaatttgct
     5221 aaaggtactg acttcgttgt tgtcagagaa ttagtgggag gtatttactt tggtaagaga
     5281 aaggaagacg atggtgatgg tgtcgcttgg gatagtgaac aatacaccgt tccagaagtg
     5341 caaagaatca caagaatggc cgctttcatg gccctacaac atgagccacc attgcctatt
     5401 tggtccttgg ataaagctaa tgttttggcc tcttcaagat tatggagaaa aactgtggag
     5461 gaaaccatca agaacgaatt tcctacattg aaggttcaac atcaattgat tgattctgcc
     5521 gccatgatcc tagttaagaa cccaacccac ctaaatggta ttataatcac cagcaacatg
     5581 tttggtgata tcatctccga tgaagcctcc gttatcccag gttccttggg tttgttgcca
     5641 tctgcgtcct tggcctcttt gccagacaag aacaccgcat ttggtttgta cgaaccatgc
     5701 cacggttctg ctccagattt gccaaagaat aaggtcaacc ctatcgccac tatcttgtct
     5761 gctgcaatga tgttgaaatt gtcattgaac ttgcctgaag aaggtaaggc cattgaagat
     5821 gcagttaaaa aggttttgga tgcaggtatc agaactggtg atttaggtgg ttccaacagt
     5881 accaccgaag tcggtgatgc tgtcgccgaa gaagttaaga aaatccttg
//
