LOCUS       sequence-217503-        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, 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 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"; doi: 
            "10.1083/jcb.201807195"; journalName: "J Cell Biol"; date: 
            "2019-05-6- 6"; volume: "218"; issue: "5"; pages: "1582-1601"
COMMENT     Sequence Label: YIplac128-pOst1-ESCargo
FEATURES             Location/Qualifiers
     source          1..5927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..29)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(183..771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(263..282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(942..1733,1734..1802))
                     /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     1565..1584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1803..1907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1975..1993
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1995..2399
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2400..3494
                     /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"
     primer_bind     complement(3635..3657)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3757..3776
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3900..4330
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             4425..5096
                     /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(4602..4622)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     4820..4839
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5014..5037
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             5109..5429
                     /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      5443..5690
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5459..5477)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5709..5725)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5709..5725)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5722..5744)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5733..5749
                     /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(5757..5763,5764..5781,5782..5787))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5802..5823
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
       61 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      181 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      601 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
      661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      721 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1081 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1801 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1861 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1921 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     1981 cgtatcacga ggccaactgt gggaatactc aggtatcgta agatgcaaga gttcgaatct
     2041 cttagcaacc attatttttt tcctcaacat aacgagaaca cacaggggcg ctatcgcaca
     2101 gaatcaaatt cgatgactgg aaattttttg ttaatttcag aggtcgcctg acgcatatac
     2161 ctttttcaac tgaaaaattg ggagaaaaag gaaaggtgag aggccggaac cggcttttca
     2221 tatagaatag agaagcgttc atgactaaat gcttgcatca caatacttga agttgacaat
     2281 attatttaag gacctattgt tttttccaat aggtggttag caatcgtctt actttctaac
     2341 ttttcttacc ttttacattt cagcaatata tatatatatt tcaaggatat accattctaa
     2401 tgtctgcccc taagaagatc gtcgttttgc caggtgacca cgttggtcaa gaaatcacag
     2461 ccgaagccat taaggttctt aaagctattt ctgatgttcg ttccaatgtc aagttcgatt
     2521 tcgaaaatca tttaattggt ggtgctgcta tcgatgctac aggtgtccca cttccagatg
     2581 aggcgctgga agcctccaag aaggttgatg ccgttttgtt aggtgctgtg ggtggtccta
     2641 aatggggtac aggtagtgtt agacctgaac aaggtttact aaaaatccgt aaagaacttc
     2701 aattgtacgc caacttaaga ccatgtaact ttgcatccga ctctctttta gacttatctc
     2761 caatcaagcc acaatttgct aaaggtactg acttcgttgt tgtcagagaa ttagtgggag
     2821 gtatttactt tggtaagaga aaggaagacg atggtgatgg tgtcgcttgg gatagtgaac
     2881 aatacaccgt tccagaagtg caaagaatca caagaatggc cgctttcatg gccctacaac
     2941 atgagccacc attgcctatt tggtccttgg ataaagctaa tgttttggcc tcttcaagat
     3001 tatggagaaa aactgtggag gaaaccatca agaacgaatt tcctacattg aaggttcaac
     3061 atcaattgat tgattctgcc gccatgatcc tagttaagaa cccaacccac ctaaatggta
     3121 ttataatcac cagcaacatg tttggtgata tcatctccga tgaagcctcc gttatcccag
     3181 gttccttggg tttgttgcca tctgcgtcct tggcctcttt gccagacaag aacaccgcat
     3241 ttggtttgta cgaaccatgc cacggttctg ctccagattt gccaaagaat aaggttgacc
     3301 ctatcgccac tatcttgtct gctgcaatga tgttgaaatt gtcattgaac ttgcctgaag
     3361 aaggtaaggc cattgaagat gcagttaaaa aggttttgga tgcaggtatc agaactggtg
     3421 atttaggtgg ttccaacagt accaccgaag tcggtgatgc tgtcgccgaa gaagttaaga
     3481 aaatccttgc ttaaaaagat tctctttttt tatgatattt gtacataaac tttataaatg
     3541 aaattcataa tagaaacgac acgaaattac aaaatggaat atgttcatag ggtaggccct
     3601 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     3661 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     3721 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
     3781 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     3841 ggcggacctt aatacattca gacacttctg cggtatcacc ctacttattc ccttcgagat
     3901 tatatctagg aacccatcag gttggtggaa gattacccgt tctaagactt ttcagcttcc
     3961 tctattgatg ttacacctgg acaccccttt tctggcatcc agtttttaat cttcagtggc
     4021 atgtgagatt ctccgaaatt aattaaagca atcacacaat tctctcggat accacctcgg
     4081 ttgaaactga caggtggttt gttacgcatg ctaatgcaaa ggagcctata tacctttggc
     4141 tcggctgctg taacagggaa tataaagggc agcataattt aggagtttag tgaacttgca
     4201 acatttacta ttttcccttc ttacgtaaat atttttcttt ttaattctaa atcaatcttt
     4261 ttcaattttt tgtttgtatt cttttcttgc ttaaatctat aactacaaaa aacacataca
     4321 taaactaaaa gaattcaaaa atgaggcagg tttggttctc ttggattgtg ggattgttcc
     4381 tatgtttttt caacgtgtct tctgctgcgc cggtgaacac caccgatagc actgagaacg
     4441 tcatcaagcc cttcatgcgc ttcaaggtgc acatggaggg ctcagtaaat ggtcatgagt
     4501 tcgagatcga gggcgagggc gagggcaagc cctacgaggg cacccagacc gccaagctgc
     4561 aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc cagttccagt
     4621 acggctccaa ggtgtacgtg aagcaccccg ccgacatccc cgactacaag aagctgtcct
     4681 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
     4741 tgacccagga ctcctccctg caggacggca ccttcatcta ccacgtgaag ttcatcggcg
     4801 tgaacttccc ctccgacggc cccgtaatgc agaagaagac tctgggctgg gagccctcca
     4861 ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccacaag gcgctgaagc
     4921 tgaagggcgg cggccactac ctggtggagt tcaagtcaat ctacatggcc aagaagcccg
     4981 tgaagctgcc cggctactac tacgtggact ccaagctgga catcacctcc cacaacgagg
     5041 actacaccgt ggtggagcag tacgagcgcg ccgaggcccg ccaccacctg ttccagggat
     5101 cctctagagg agtgcaggtg gaaaccatct ccccaggaga cgggcgcacc ttccccaagc
     5161 gcggccagac cgttgtggtg cactacaccg ggatgcttga agatggaaag aaactagatt
     5221 cctcccggga cagaaacaag ccctttaagt ttatgctagg caagcaggag gtgatccgag
     5281 gctgggaaga aggggttgcc cagatgagtg tgggtcagag agccaaactg actatatctc
     5341 cagattatgc ctatggtgcc actgggcacc caggcgttat cccaccacat gccactctcg
     5401 tcttcgatgt ggagcttcta aaactggaat aaactagagt catcatgtaa ttagttatgt
     5461 cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac
     5521 aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat
     5581 ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac
     5641 tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc aagcttggcg
     5701 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5761 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5821 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5881 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcg
//
