LOCUS       sequence-382339-        8668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EMC1-GFP-APEX2 chimera for labeling yeast endoplasmic 
            reticulum (ER). Uses auxotrophic marker URA3(Kluyveromyces lactis). 
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8668)
  AUTHORS   Li D, Yang SG, He CW, Zhang ZT, Liang Y, Li H, Zhu J, Su X, Gong Q, 
            Xie Z
  TITLE     Excess diacylglycerol at the endoplasmic reticulum disrupts 
            endomembrane homeostasis and autophagy.
  JOURNAL   BMC Biol. 2020 Aug 28;18(1):107. doi: 10.1186/s12915-020-00837-w.
  PUBMED    32859196
REFERENCE   2  (bases 1 to 8668)
  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: "BMC Biol.";
            date: "2020-08-28"; volume: "18(1):107. doi"; pages: " 
            10.1186/s12915-020-00837-w"
COMMENT     Sequence Label: pTPI1-EMC1-GFP-APEX2-URA3
FEATURES             Location/Qualifiers
     source          1..8668
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(143..731)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(223..242)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(902..1693,1694..1762))
                     /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     1525..1544
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1763..1867)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1935..1953
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1991..2013)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2113..2132
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2213..2231
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2213..2230
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        2255..2774
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             5106..5819
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             5106..5819
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             5862..6608
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     CDS             6615..6641
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      6724..6911
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(6779..6803)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    6963..6996
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    8448..8481
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(8534..8553)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8535..8553)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8640..8657)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
       61 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
      121 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
      181 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
      241 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
      301 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
      361 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
      421 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
      481 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
      541 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
      601 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
      661 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
      721 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
      781 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
      841 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
      901 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
      961 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     1021 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     1081 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     1141 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     1201 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     1261 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     1321 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     1381 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     1441 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     1501 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     1561 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     1621 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     1681 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     1741 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     1801 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     1861 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     1921 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     1981 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     2041 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     2101 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg gacatattgt
     2161 cgttagaacg cggctacaat taatacataa ccttatgtat catacacata cgatttaggt
     2221 gacactatag aacgcggccg ccagctgaag cttccctacg ttagtgtgag cgggatttaa
     2281 actgtgagga ccttaataca ttcagacact tctgcggtat caccctactt attcccttcg
     2341 agattatatc taggaaccca tcaggttggt ggaagattac ccgttctaag acttttcagc
     2401 ttcctctatt gatgttacac ctggacaccc cttttctggc atccagtttt taatcttcag
     2461 tggcatgtga gattctccga aattaattaa agcaatcaca caattctctc ggataccacc
     2521 tcggttgaaa ctgacaggtg gtttgttacg catgctaatg caaaggagcc tatatacctt
     2581 tggctcggct gctgtaacag ggaatataaa gggcagcata atttaggagt ttagtgaact
     2641 tgcaacattt actattttcc cttcttacgt aaatattttt ctttttaatt ctaaatcaat
     2701 ctttttcaat tttttgtttg tattcttttc ttgcttaaat ctataactac aaaaaacaca
     2761 tacataaact aaaaatgaag ataacgtgta cagacttggt gtacgtcttc attttactct
     2821 tcctaaacac gagttgtgtc caagccgttt tttcagatga tgcatttatc actgattggc
     2881 aactggctaa cttaggtcct tgggagaaag tcatccctga ttctcgagac cgcaacaggg
     2941 ttctcatctt atcgaaccct accgaaactt cctgcttagt ttcttcgttt aacgtttctt
     3001 ccggacagat tcttttcaga aacgttttac cctttaccat tgatgagatt caactggata
     3061 gtaatgacca taacgcaatg gtttgtgtga actcttcaag caaccattgg cagaaatatg
     3121 atttacacga ttggttttta ctagaggaag gcgtagataa tgccccttct acgaccattt
     3181 tacctcaatc ctcatattta aacgatcaag tatctattaa gaacaatgaa ctacatattc
     3241 tcgatgagca gtcaaaactg gcagaatgga aattggagtt acctcaaggg ttcaataaag
     3301 tggaatattt tcatcgtgaa gatcccctgg cgttagtgtt gaacgttaat gatacccaat
     3361 atatgggatt ctctgccaat ggcacagaat tgatccccgt ttggcaaaga gatgaatggt
     3421 tgactaacgt ggtagactat gctgtattgg acgtcttcga ttctagggat gtggagttga
     3481 acaaagatat gaaagcggaa cttgattcaa attcgctttg gaatgcttac tggcttagat
     3541 tgacaactaa ttggaatcgc cttatcaact tattgaaaga aaaccaattc tcaccaggac
     3601 gtgtcttcac taaactccta gctctagacg ctaaggatac cacggtatca gatttgaagt
     3661 tcggattcgc caaaatctta attgttttga cgcatgatgg ctttatcggc ggccttgata
     3721 tggtcaataa gggccaactt atctggaaac tcgatttaga aattgatcag ggcgtcaaaa
     3781 tgttctggac ggataaaaac catgacgaac ttgttgtttt ttcgcatgat gggcattatt
     3841 tgacaattga agttactaaa gatcaaccga ttatcaaatc aagatccccc ctatctgaaa
     3901 ggaaaactgt tgattccgtt attaggctga atgaacatga tcaccagtat ctgattaagt
     3961 ttgaggataa ggatcattta ctgttcaaat tgaatcccgg caagaatacg gatgtaccaa
     4021 tagttgccaa caaccattct agttcccaca tattcgtcac agagcatgac acgaatggca
     4081 tttatggcta cataatcgaa aacgatacgg taaaacaaac ttggaaaaaa gccgtaaatt
     4141 cgaaagagaa aatggtggca tatagcaaga gggaaacaac aaacctaaac actcttggta
     4201 ttacactagg tgacaaatcg gttctttata aatatttgta ccccaaccta gcggcttatc
     4261 tgatcgctaa tgaagaacat catacaatca cttttaactt aattgatacc attacaggag
     4321 aaatcctcat tacccaagag cacaaggatt ctccggattt taggtttcca atggatattg
     4381 ttttcggtga atattgggtc gtttattcct atttcagttc tgaacctgtt ccagaacaaa
     4441 agttagtagt ggtggaatta tatgagtcac taaccccaga tgagcgtttg tctaactcaa
     4501 gcgacaattt ttcttatgat ccattgactg gacacattaa caaacctcaa tttcaaacta
     4561 aacaattcat ttttcccgag attatcaaaa caatgtccat ttccaagaca acggatgata
     4621 ttaccacaaa ggcaatcgtt atggaattag aaaatggaca aatcacctac ataccaaagc
     4681 ttttattgaa tgcaagaggt aaaccagcag aagaaatggc caaggataag aaaaaagagt
     4741 ttatggctac cccatacacg ccagttatcc caattaatga taatttcatt atcactcatt
     4801 tcagaaatct attgccagga tccgattcgc agttgatctc catcccaacc aatctggaat
     4861 ccacaagcat tatatgtgat ctaggccttg atgtattttg tacaaggatc acaccttcgg
     4921 gccaatttga tttaatgagt cctactttcg aaaagggtaa attgcttatt actatattcg
     4981 tcttgttggt gatcacgtat tttatccgtc cttctgtttc aaacaagaag ttgaaatccc
     5041 aatggctaat taaagcttcg tacgctgcag gtcgacggat cggtgacggt gctggtttaa
     5101 ttaacatgtc taaaggtgaa gaattattca ctggtgttgt cccaattttg gttgaattag
     5161 atggtgatgt taatggtcac aaattttctg tctccggtga aggtgaaggt gatgctactt
     5221 acggtaaatt gaccttaaaa tttatttgta ctactggtaa attgccagtt ccatggccaa
     5281 ccttagtcac tactttaact tatggtgttc aatgtttttc tagataccca gatcatatga
     5341 aacaacatga ctttttcaag tctgccatgc cagaaggtta tgttcaagaa agaactattt
     5401 ttttcaaaga tgacggtaac tacaagacca gagctgaagt caagtttgaa ggtgatacct
     5461 tagttaatag aatcgaatta aaaggtattg attttaaaga agatggtaac attttaggtc
     5521 acaaattgga atacaactat aactctcaca atgtttacat catggctgac aaacaaaaga
     5581 atggtatcaa agttaacttc aaaattagac acaacattga agatggttct gttcaattag
     5641 ctgaccatta tcaacaaaat actccaattg gtgatggtcc agtcttgtta ccagacaacc
     5701 attacttatc cactcaatct gccttatcca aagatccaaa cgaaaagaga gaccacatgg
     5761 tcttgttaga atttgttact gctgctggta ttacccatgg tatggatgaa ttgtacaaag
     5821 ctgcaggtcg tgggattcct gggttaatta acatcgctag cggaaagtct tacccaactg
     5881 tgagtgctga ttaccaggac gccgttgaga aggcgaagaa gaagctcaga ggcttcatcg
     5941 ctgagaagag atgcgctcct ctaatgctcc gtttggcatt ccactctgct ggaacctttg
     6001 acaagggcac gaagaccggt ggacccttcg gaaccatcaa gcaccctgcc gaactggctc
     6061 acagcgctaa caacggtctt gacatcgctg ttaggctttt ggagccactc aaggcggagt
     6121 tccctatttt gagctacgcc gatttctacc agttggctgg cgttgttgcc gttgaggtca
     6181 cgggtggacc taaggttcca ttccaccctg gaagagagga caagcctgag ccaccaccag
     6241 agggtcgctt gcccgatccc actaagggtt ctgaccattt gagagatgtg tttggcaaag
     6301 ctatggggct tactgaccaa gatatcgttg ctctatctgg gggtcacact attggagctg
     6361 cacacaagga gcgttctgga tttgagggtc cctggacctc taatcctctt attttcgaca
     6421 actcatactt cacggagttg ttgagtggtg agaaggaagg tctccttcag ctaccttctg
     6481 acaaggctct tttgtctgac cctgtattcc gccctctcgt tgacaaatat gcagcggacg
     6541 aagatgcctt ctttgctgat tacgctgagg ctcaccaaaa gctttccgag cttgggtttg
     6601 ctgatgccct gcagctgcct cccctggagc gcctgaccct ggacgctcag tgcagatctt
     6661 agcctaggtg aggcgcgctc agtgcagatc ttagcctagg tgaggcgcgc cacttctaaa
     6721 taagcgaatt tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt
     6781 gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac
     6841 tctttcctgt aggtcaggtt gctttctcag gtatagtatg aggtcgctct tattgaccac
     6901 acctctaccg gcagatccgc tagggataac agggtaatat ctgcaggtcg acaaccctta
     6961 atataacttc gtataatgta tgctatacga agttattagg tctagagatc ccaatacaac
     7021 agatcacgtg atctttttgt aagatgaagt tgaagtagtg ttgcaccgtg ccaatgcagg
     7081 tggctattag attaaatatg tgatttgttc tattaagttt cctgtataat taatggggag
     7141 cgctgattct cttttggtac gcttcccatc cagcatttct gtatctttca ccttcaacct
     7201 taggatctct acccttggcg aaaagtcctc tgccaacaat gatgatatct gatccaccac
     7261 ttacaacttc gtcgacggtt ctgtactgct gacccaatgc atcgcctttg tcgtctaaac
     7321 ctacacctgg ggtcatgatt agccaatcaa acccttcttc tcttcctccc atatcgttct
     7381 gagcaatgaa cccaataacg aaatctttat cactctttgc aatatcaacg gtacccttag
     7441 tatattcacc gtgtgctaga gaacccttgg aagacaattc agcaagcatc aataatcccc
     7501 ttggttcttt ggtgacctct tgcgcacctt gtttcaagcc agcaacaata ccagcaccag
     7561 taaccccgtg ggcgttggtg atatcagacc attctgcgat acggtaaacg cccgatgtat
     7621 attgtaattt gactgtgtta ccgatatcgg cgaattttct gtcctcaaat atcaagaact
     7681 tgtatttctc tgccaatgct ttcaatggaa cgacagtacc ctcataactg aaatcatcca
     7741 agatatcaac gtgtgttttc aaaaggcaaa tgtatggacc caacgtttca acaagtttca
     7801 atagctcatc agtcgaacga acgtcaagag aagcacacaa attggtcttc ttttcatcca
     7861 ttaaacgtaa aagtttcgat gcaaccggac ttgcatgagt ctcagctcta ctggtatatg
     7921 attttgtgga catggtgcaa ctaattgacg ggagtgtatt gacgctggcg tactggcttt
     7981 cacaaaatgg cccaatcaca accacatctt agatagttga aatgacttta gataacatca
     8041 attgagatga gcttaatcat gtcaaagcta aaagtgtcac catgaacgac aattcttaag
     8101 caaatcacgt gatatagatc cacgaataac caccatttga tgctcgaggc aagtaatgtg
     8161 tgtaaaaaaa tgcgttacca ccatccaatg cagaccgatc ttctacccag aatcacatat
     8221 atttatgtac cgagtacctt ttttctatct tccaattgct tctcccatat gattgtctcc
     8281 gtaagctcga aatttctaag ttggatttta atcttcacgc aggatgacag ttcgatgagc
     8341 ttctgaggag tgtttagaac ataatcattt atccatggtc tatctcttct tgtcgctttt
     8401 tctcctcgat agaacctaaa taaaacgagc tctcgagaac ccttaatata acttcgtata
     8461 atgtatgcta tacgaagtta ttaggtgata tcagatccac tagtggccta tgcggccgcg
     8521 gatctgccgg tctccctata gtgagtcgta ttaatttcga taagccaggt taacctgcat
     8581 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     8641 tcgctcactg actcgctgcg ctcggtcg
//
