LOCUS       sequence-422281-        8668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing Emc1-GFP-APEX2. GFP interferes with APEX staining..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8668)
  AUTHORS   Li H, He CW, Zhu J, Xie Z
  TITLE     A Validated Set of Ascorbate Peroxidase-Based Organelle Markers for 
            Electron Microscopy of Saccharomyces cerevisiae.
  JOURNAL   mSphere. 2022 Aug 31;7(4):e0010722. doi: 10.1128/msphere.00107-22. 
            Epub 2022 Jun 21.
  PUBMED    35727034
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: "mSphere."; 
            date: "2022-08-31"; volume: "7(4):e0010722. doi"; pages: " 
            10.1128/msphere.00107-22. Epub 2022 Jun 21"
COMMENT     Sequence Label: ClhN-pTPI1-Emc1-GFP-APEX2-Ura
FEATURES             Location/Qualifiers
     source          1..8668
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        26..545
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             2877..3590
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2877..3590
                     /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             3633..4379
                     /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             4386..4412
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      4495..4682
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4550..4574)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    4734..4767
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    6219..6252
                     /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(6305..6324)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6306..6324)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6411..6428)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6582..7170)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6662..6681)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7341..8132,8133..8201))
                     /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     7964..7983
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8202..8306)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8374..8392
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8430..8452)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8552..8571
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(8652..8668,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(8652..8668,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttccctac gttagtgtga gcgggattta aactgtgagg
       61 accttaatac attcagacac ttctgcggta tcaccctact tattcccttc gagattatat
      121 ctaggaaccc atcaggttgg tggaagatta cccgttctaa gacttttcag cttcctctat
      181 tgatgttaca cctggacacc ccttttctgg catccagttt ttaatcttca gtggcatgtg
      241 agattctccg aaattaatta aagcaatcac acaattctct cggataccac ctcggttgaa
      301 actgacaggt ggtttgttac gcatgctaat gcaaaggagc ctatatacct ttggctcggc
      361 tgctgtaaca gggaatataa agggcagcat aatttaggag tttagtgaac ttgcaacatt
      421 tactattttc ccttcttacg taaatatttt tctttttaat tctaaatcaa tctttttcaa
      481 ttttttgttt gtattctttt cttgcttaaa tctataacta caaaaaacac atacataaac
      541 taaaaatgaa gataacgtgt acagacttgg tgtacgtctt cattttactc ttcctaaaca
      601 cgagttgtgt ccaagccgtt ttttcagatg atgcatttat cactgattgg caactggcta
      661 acttaggtcc ttgggagaaa gtcatccctg attctcgaga ccgcaacagg gttctcatct
      721 tatcgaaccc taccgaaact tcctgcttag tttcttcgtt taacgtttct tccggacaga
      781 ttcttttcag aaacgtttta ccctttacca ttgatgagat tcaactggat agtaatgacc
      841 ataacgcaat ggtttgtgtg aactcttcaa gcaaccattg gcagaaatat gatttacacg
      901 attggttttt actagaggaa ggcgtagata atgccccttc tacgaccatt ttacctcaat
      961 cctcatattt aaacgatcaa gtatctatta agaacaatga actacatatt ctcgatgagc
     1021 agtcaaaact ggcagaatgg aaattggagt tacctcaagg gttcaataaa gtggaatatt
     1081 ttcatcgtga agatcccctg gcgttagtgt tgaacgttaa tgatacccaa tatatgggat
     1141 tctctgccaa tggcacagaa ttgatccccg tttggcaaag agatgaatgg ttgactaacg
     1201 tggtagacta tgctgtattg gacgtcttcg attctaggga tgtggagttg aacaaagata
     1261 tgaaagcgga acttgattca aattcgcttt ggaatgctta ctggcttaga ttgacaacta
     1321 attggaatcg ccttatcaac ttattgaaag aaaaccaatt ctcaccagga cgtgtcttca
     1381 ctaaactcct agctctagac gctaaggata ccacggtatc agatttgaag ttcggattcg
     1441 ccaaaatctt aattgttttg acgcatgatg gctttatcgg cggccttgat atggtcaata
     1501 agggccaact tatctggaaa ctcgatttag aaattgatca gggcgtcaaa atgttctgga
     1561 cggataaaaa ccatgacgaa cttgttgttt tttcgcatga tgggcattat ttgacaattg
     1621 aagttactaa agatcaaccg attatcaaat caagatcccc cctatctgaa aggaaaactg
     1681 ttgattccgt tattaggctg aatgaacatg atcaccagta tctgattaag tttgaggata
     1741 aggatcattt actgttcaaa ttgaatcccg gcaagaatac ggatgtacca atagttgcca
     1801 acaaccattc tagttcccac atattcgtca cagagcatga cacgaatggc atttatggct
     1861 acataatcga aaacgatacg gtaaaacaaa cttggaaaaa agccgtaaat tcgaaagaga
     1921 aaatggtggc atatagcaag agggaaacaa caaacctaaa cactcttggt attacactag
     1981 gtgacaaatc ggttctttat aaatatttgt accccaacct agcggcttat ctgatcgcta
     2041 atgaagaaca tcatacaatc acttttaact taattgatac cattacagga gaaatcctca
     2101 ttacccaaga gcacaaggat tctccggatt ttaggtttcc aatggatatt gttttcggtg
     2161 aatattgggt cgtttattcc tatttcagtt ctgaacctgt tccagaacaa aagttagtag
     2221 tggtggaatt atatgagtca ctaaccccag atgagcgttt gtctaactca agcgacaatt
     2281 tttcttatga tccattgact ggacacatta acaaacctca atttcaaact aaacaattca
     2341 tttttcccga gattatcaaa acaatgtcca tttccaagac aacggatgat attaccacaa
     2401 aggcaatcgt tatggaatta gaaaatggac aaatcaccta cataccaaag cttttattga
     2461 atgcaagagg taaaccagca gaagaaatgg ccaaggataa gaaaaaagag tttatggcta
     2521 ccccatacac gccagttatc ccaattaatg ataatttcat tatcactcat ttcagaaatc
     2581 tattgccagg atccgattcg cagttgatct ccatcccaac caatctggaa tccacaagca
     2641 ttatatgtga tctaggcctt gatgtatttt gtacaaggat cacaccttcg ggccaatttg
     2701 atttaatgag tcctactttc gaaaagggta aattgcttat tactatattc gtcttgttgg
     2761 tgatcacgta ttttatccgt ccttctgttt caaacaagaa gttgaaatcc caatggctaa
     2821 ttaaagcttc gtacgctgca ggtcgacgga tcggtgacgg tgctggttta attaacatgt
     2881 ctaaaggtga agaattattc actggtgttg tcccaatttt ggttgaatta gatggtgatg
     2941 ttaatggtca caaattttct gtctccggtg aaggtgaagg tgatgctact tacggtaaat
     3001 tgaccttaaa atttatttgt actactggta aattgccagt tccatggcca accttagtca
     3061 ctactttaac ttatggtgtt caatgttttt ctagataccc agatcatatg aaacaacatg
     3121 actttttcaa gtctgccatg ccagaaggtt atgttcaaga aagaactatt tttttcaaag
     3181 atgacggtaa ctacaagacc agagctgaag tcaagtttga aggtgatacc ttagttaata
     3241 gaatcgaatt aaaaggtatt gattttaaag aagatggtaa cattttaggt cacaaattgg
     3301 aatacaacta taactctcac aatgtttaca tcatggctga caaacaaaag aatggtatca
     3361 aagttaactt caaaattaga cacaacattg aagatggttc tgttcaatta gctgaccatt
     3421 atcaacaaaa tactccaatt ggtgatggtc cagtcttgtt accagacaac cattacttat
     3481 ccactcaatc tgccttatcc aaagatccaa acgaaaagag agaccacatg gtcttgttag
     3541 aatttgttac tgctgctggt attacccatg gtatggatga attgtacaaa gctgcaggtc
     3601 gtgggattcc tgggttaatt aacatcgcta gcggaaagtc ttacccaact gtgagtgctg
     3661 attaccagga cgccgttgag aaggcgaaga agaagctcag aggcttcatc gctgagaaga
     3721 gatgcgctcc tctaatgctc cgtttggcat tccactctgc tggaaccttt gacaagggca
     3781 cgaagaccgg tggacccttc ggaaccatca agcaccctgc cgaactggct cacagcgcta
     3841 acaacggtct tgacatcgct gttaggcttt tggagccact caaggcggag ttccctattt
     3901 tgagctacgc cgatttctac cagttggctg gcgttgttgc cgttgaggtc acgggtggac
     3961 ctaaggttcc attccaccct ggaagagagg acaagcctga gccaccacca gagggtcgct
     4021 tgcccgatcc cactaagggt tctgaccatt tgagagatgt gtttggcaaa gctatggggc
     4081 ttactgacca agatatcgtt gctctatctg ggggtcacac tattggagct gcacacaagg
     4141 agcgttctgg atttgagggt ccctggacct ctaatcctct tattttcgac aactcatact
     4201 tcacggagtt gttgagtggt gagaaggaag gtctccttca gctaccttct gacaaggctc
     4261 ttttgtctga ccctgtattc cgccctctcg ttgacaaata tgcagcggac gaagatgcct
     4321 tctttgctga ttacgctgag gctcaccaaa agctttccga gcttgggttt gctgatgccc
     4381 tgcagctgcc tcccctggag cgcctgaccc tggacgctca gtgcagatct tagcctaggt
     4441 gaggcgcgct cagtgcagat cttagcctag gtgaggcgcg ccacttctaa ataagcgaat
     4501 ttcttatgat ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa
     4561 ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg
     4621 taggtcaggt tgctttctca ggtatagtat gaggtcgctc ttattgacca cacctctacc
     4681 ggcagatccg ctagggataa cagggtaata tctgcaggtc gacaaccctt aatataactt
     4741 cgtataatgt atgctatacg aagttattag gtctagagat cccaatacaa cagatcacgt
     4801 gatctttttg taagatgaag ttgaagtagt gttgcaccgt gccaatgcag gtggctatta
     4861 gattaaatat gtgatttgtt ctattaagtt tcctgtataa ttaatgggga gcgctgattc
     4921 tcttttggta cgcttcccat ccagcatttc tgtatctttc accttcaacc ttaggatctc
     4981 tacccttggc gaaaagtcct ctgccaacaa tgatgatatc tgatccacca cttacaactt
     5041 cgtcgacggt tctgtactgc tgacccaatg catcgccttt gtcgtctaaa cctacacctg
     5101 gggtcatgat tagccaatca aacccttctt ctcttcctcc catatcgttc tgagcaatga
     5161 acccaataac gaaatcttta tcactctttg caatatcaac ggtaccctta gtatattcac
     5221 cgtgtgctag agaacccttg gaagacaatt cagcaagcat caataatccc cttggttctt
     5281 tggtgacctc ttgcgcacct tgtttcaagc cagcaacaat accagcacca gtaaccccgt
     5341 gggcgttggt gatatcagac cattctgcga tacggtaaac gcccgatgta tattgtaatt
     5401 tgactgtgtt accgatatcg gcgaattttc tgtcctcaaa tatcaagaac ttgtatttct
     5461 ctgccaatgc tttcaatgga acgacagtac cctcataact gaaatcatcc aagatatcaa
     5521 cgtgtgtttt caaaaggcaa atgtatggac ccaacgtttc aacaagtttc aatagctcat
     5581 cagtcgaacg aacgtcaaga gaagcacaca aattggtctt cttttcatcc attaaacgta
     5641 aaagtttcga tgcaaccgga cttgcatgag tctcagctct actggtatat gattttgtgg
     5701 acatggtgca actaattgac gggagtgtat tgacgctggc gtactggctt tcacaaaatg
     5761 gcccaatcac aaccacatct tagatagttg aaatgacttt agataacatc aattgagatg
     5821 agcttaatca tgtcaaagct aaaagtgtca ccatgaacga caattcttaa gcaaatcacg
     5881 tgatatagat ccacgaataa ccaccatttg atgctcgagg caagtaatgt gtgtaaaaaa
     5941 atgcgttacc accatccaat gcagaccgat cttctaccca gaatcacata tatttatgta
     6001 ccgagtacct tttttctatc ttccaattgc ttctcccata tgattgtctc cgtaagctcg
     6061 aaatttctaa gttggatttt aatcttcacg caggatgaca gttcgatgag cttctgagga
     6121 gtgtttagaa cataatcatt tatccatggt ctatctcttc ttgtcgcttt ttctcctcga
     6181 tagaacctaa ataaaacgag ctctcgagaa cccttaatat aacttcgtat aatgtatgct
     6241 atacgaagtt attaggtgat atcagatcca ctagtggcct atgcggccgc ggatctgccg
     6301 gtctccctat agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc
     6361 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     6421 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     6481 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     6541 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     6601 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6661 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6721 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6781 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6841 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6901 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6961 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     7021 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     7081 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     7141 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     7201 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     7261 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     7321 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     7381 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     7441 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     7501 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     7561 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     7621 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     7681 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     7741 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     7801 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     7861 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     7921 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     7981 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     8041 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     8101 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     8161 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     8221 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     8281 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     8341 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     8401 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     8461 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     8521 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
     8581 caccatatgg acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc
     8641 atacacatac gatttaggtg acactata
//
