LOCUS       sequence-422271-        6794 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing Cox4-GFP-APEX2. GFP interferes with APEX staining. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6794)
  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 6794)
  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-Cox4-GFP-APEX2-Ura
FEATURES             Location/Qualifiers
     source          1..6794
                     /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             546..611
                     /codon_start=1
                     /product="mitochondrial presequence of yeast cytochrome c 
                     oxidase subunit IV (Hurt et al., 1984)"
                     /label=pre-CoxIV
                     /translation="MLSLRQSIRFFKPATRTLCSSR"
     CDS             1035..1748
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             1035..1748
                     /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             1791..2537
                     /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             2544..2570
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      2621..2808
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2676..2700)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    2860..2893
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4345..4378
                     /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(4431..4450)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4432..4450)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4537..4554)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4708..5296)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4788..4807)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5467..6258,6259..6327))
                     /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     6090..6109
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6328..6432)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6500..6518
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6556..6578)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6678..6697
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6778..6794,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6778..6794,1)
                     /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 taaaaatgct ttcactacgt caatctataa gatttttcaa gccagccaca agaactttgt
      601 gtagctctag atatctgctt cagcaaaaac ccgtggtgaa aactgcccaa aacttagcag
      661 aagttaatgg tccagaaact ttgattggtc ctggtgctaa agagggtacc gttccaacag
      721 acctagatca agaaactggt ttagctaggt tagaattatt gggtaaatta gagggtatcg
      781 atgttttcga caccaaacca ttagattcgt ccaggaaggg taccatgaaa gatccgatca
      841 tcattgaatc ttatgatgat tatcgttatg tcggttgtac gggttctccc gctggttcac
      901 atactattat gtggttaaaa ccaactgtta acgaagtcgc cagatgctgg gaatgtggtt
      961 ctgtttacaa actaaaccct gttggtgttc caaatgatga ccaccatcac ctttctcttg
     1021 aagttgctga agaaatgtct aaaggtgaag aattattcac tggtgttgtc ccaattttgg
     1081 ttgaattaga tggtgatgtt aatggtcaca aattttctgt ctccggtgaa ggtgaaggtg
     1141 atgctactta cggtaaattg accttaaaat ttatttgtac tactggtaaa ttgccagttc
     1201 catggccaac cttagtcact actttaactt atggtgttca atgtttttct agatacccag
     1261 atcatatgaa acaacatgac tttttcaagt ctgccatgcc agaaggttat gttcaagaaa
     1321 gaactatttt tttcaaagat gacggtaact acaagaccag agctgaagtc aagtttgaag
     1381 gtgatacctt agttaataga atcgaattaa aaggtattga ttttaaagaa gatggtaaca
     1441 ttttaggtca caaattggaa tacaactata actctcacaa tgtttacatc atggctgaca
     1501 aacaaaagaa tggtatcaaa gttaacttca aaattagaca caacattgaa gatggttctg
     1561 ttcaattagc tgaccattat caacaaaata ctccaattgg tgatggtcca gtcttgttac
     1621 cagacaacca ttacttatcc actcaatctg ccttatccaa agatccaaac gaaaagagag
     1681 accacatggt cttgttagaa tttgttactg ctgctggtat tacccatggt atggatgaat
     1741 tgtacaaagc tgcaggtcgt gggattcctg ggttaattaa catcgctagc ggaaagtctt
     1801 acccaactgt gagtgctgat taccaggacg ccgttgagaa ggcgaagaag aagctcagag
     1861 gcttcatcgc tgagaagaga tgcgctcctc taatgctccg tttggcattc cactctgctg
     1921 gaacctttga caagggcacg aagaccggtg gacccttcgg aaccatcaag caccctgccg
     1981 aactggctca cagcgctaac aacggtcttg acatcgctgt taggcttttg gagccactca
     2041 aggcggagtt ccctattttg agctacgccg atttctacca gttggctggc gttgttgccg
     2101 ttgaggtcac gggtggacct aaggttccat tccaccctgg aagagaggac aagcctgagc
     2161 caccaccaga gggtcgcttg cccgatccca ctaagggttc tgaccatttg agagatgtgt
     2221 ttggcaaagc tatggggctt actgaccaag atatcgttgc tctatctggg ggtcacacta
     2281 ttggagctgc acacaaggag cgttctggat ttgagggtcc ctggacctct aatcctctta
     2341 ttttcgacaa ctcatacttc acggagttgt tgagtggtga gaaggaaggt ctccttcagc
     2401 taccttctga caaggctctt ttgtctgacc ctgtattccg ccctctcgtt gacaaatatg
     2461 cagcggacga agatgccttc tttgctgatt acgctgaggc tcaccaaaag ctttccgagc
     2521 ttgggtttgc tgatgccctg cagctgcctc ccctggagcg cctgaccctg gacgctcagt
     2581 gcagatctta gcctaggtga ggcgcgccac ttctaaataa gcgaatttct tatgatttat
     2641 gatttttatt attaaataag ttataaaaaa aataagtgta tacaaatttt aaagtgactc
     2701 ttaggtttta aaacgaaaat tcttattctt gagtaactct ttcctgtagg tcaggttgct
     2761 ttctcaggta tagtatgagg tcgctcttat tgaccacacc tctaccggca gatccgctag
     2821 ggataacagg gtaatatctg caggtcgaca acccttaata taacttcgta taatgtatgc
     2881 tatacgaagt tattaggtct agagatccca atacaacaga tcacgtgatc tttttgtaag
     2941 atgaagttga agtagtgttg caccgtgcca atgcaggtgg ctattagatt aaatatgtga
     3001 tttgttctat taagtttcct gtataattaa tggggagcgc tgattctctt ttggtacgct
     3061 tcccatccag catttctgta tctttcacct tcaaccttag gatctctacc cttggcgaaa
     3121 agtcctctgc caacaatgat gatatctgat ccaccactta caacttcgtc gacggttctg
     3181 tactgctgac ccaatgcatc gcctttgtcg tctaaaccta cacctggggt catgattagc
     3241 caatcaaacc cttcttctct tcctcccata tcgttctgag caatgaaccc aataacgaaa
     3301 tctttatcac tctttgcaat atcaacggta cccttagtat attcaccgtg tgctagagaa
     3361 cccttggaag acaattcagc aagcatcaat aatccccttg gttctttggt gacctcttgc
     3421 gcaccttgtt tcaagccagc aacaatacca gcaccagtaa ccccgtgggc gttggtgata
     3481 tcagaccatt ctgcgatacg gtaaacgccc gatgtatatt gtaatttgac tgtgttaccg
     3541 atatcggcga attttctgtc ctcaaatatc aagaacttgt atttctctgc caatgctttc
     3601 aatggaacga cagtaccctc ataactgaaa tcatccaaga tatcaacgtg tgttttcaaa
     3661 aggcaaatgt atggacccaa cgtttcaaca agtttcaata gctcatcagt cgaacgaacg
     3721 tcaagagaag cacacaaatt ggtcttcttt tcatccatta aacgtaaaag tttcgatgca
     3781 accggacttg catgagtctc agctctactg gtatatgatt ttgtggacat ggtgcaacta
     3841 attgacggga gtgtattgac gctggcgtac tggctttcac aaaatggccc aatcacaacc
     3901 acatcttaga tagttgaaat gactttagat aacatcaatt gagatgagct taatcatgtc
     3961 aaagctaaaa gtgtcaccat gaacgacaat tcttaagcaa atcacgtgat atagatccac
     4021 gaataaccac catttgatgc tcgaggcaag taatgtgtgt aaaaaaatgc gttaccacca
     4081 tccaatgcag accgatcttc tacccagaat cacatatatt tatgtaccga gtaccttttt
     4141 tctatcttcc aattgcttct cccatatgat tgtctccgta agctcgaaat ttctaagttg
     4201 gattttaatc ttcacgcagg atgacagttc gatgagcttc tgaggagtgt ttagaacata
     4261 atcatttatc catggtctat ctcttcttgt cgctttttct cctcgataga acctaaataa
     4321 aacgagctct cgagaaccct taatataact tcgtataatg tatgctatac gaagttatta
     4381 ggtgatatca gatccactag tggcctatgc ggccgcggat ctgccggtct ccctatagtg
     4441 agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg
     4501 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4561 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4621 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4681 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4741 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4801 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4861 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4921 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4981 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5041 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5101 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5161 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5221 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5281 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5341 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5401 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5461 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5521 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5581 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5641 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5701 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5761 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5821 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5881 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5941 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6001 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6061 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6121 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6181 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6241 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6301 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6361 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6421 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     6481 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     6541 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     6601 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     6661 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatggacat
     6721 attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt
     6781 taggtgacac tata
//
