LOCUS       sequence-424585-        7267 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 7267)
  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 7267)
  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-p1k-Cox4-GFP-APEX2-Ura
FEATURES             Location/Qualifiers
     source          1..7267
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1019..1084
                     /codon_start=1
                     /product="mitochondrial presequence of yeast cytochrome c 
                     oxidase subunit IV (Hurt et al., 1984)"
                     /label=pre-CoxIV
                     /translation="MLSLRQSIRFFKPATRTLCSSR"
     CDS             1508..2221
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             1508..2221
                     /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             2264..3010
                     /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             3017..3043
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      3094..3281
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3149..3173)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    3333..3366
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4818..4851
                     /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(4904..4923)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4905..4923)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5010..5027)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5181..5769)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5261..5280)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5940..6731,6732..6800))
                     /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     6563..6582
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6801..6905)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6973..6991
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7029..7051)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7151..7170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7251..7267,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7251..7267,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcggatt ggcccaattg ctgccaaatt tggtgaatat
       61 ggtggtgatg tcgcaatgtg gttaagtatg gcgttctcag gtgttgtata ccctccttgt
      121 agatacttag agctacgtaa atttggtcgt taattgaaat gacataagta tatattaaaa
      181 ctacggtgca gcggcaaact ttagtttaga taatattggc aggtagattc atggtatgtt
      241 aaattacgct cagggaggat atttttttgt acacattatc ttttcacatg aaaacctaag
      301 cctggtggac aattgaaatc atcgagtgat atactgacac tttgaacagt agcacaaatc
      361 gtttaatttg ctgtcacgat ctaagttttc tgtcagctga ctgtccacca ataagatcag
      421 cggattccaa aggcgtcctt cgaaaaagcg atatctccca tcttggttag tagcaaataa
      481 agggaagaga aaataagaca cactcaagga taaaatactt cgcttcttcc aagtataaac
      541 acaggaaaga aagatatacg aagattagaa tttttttcat actttttaca aaacattttt
      601 ttgtacttag gcacgctaat tgcgtaaaaa ttagcataaa caggaaataa gattattttg
      661 aaatgtatgt tataatcctc ttattgaaaa atacatgaga tgtaggagaa gaactaccag
      721 gaaagaagat atttaacaga tcgaacgaaa cggaaaagaa ctaagagaaa acaaatctgg
      781 atttgacttt aagaaaaaat ttatttgcaa gtttaactcg ttccctgctt ttctatttgg
      841 cttgagacga taattgcata attgtccaaa ttgtccatca tataatgaag aattcgatgc
      901 gaaatattcc ctgtcacttt cctttagtca acgtattctt ctccctgaag aaacagtata
      961 ctaacaatac tcacccattt cgattttgat gttgccatac aaatagataa caagcacaat
     1021 gctttcacta cgtcaatcta taagattttt caagccagcc acaagaactt tgtgtagctc
     1081 tagatatctg cttcagcaaa aacccgtggt gaaaactgcc caaaacttag cagaagttaa
     1141 tggtccagaa actttgattg gtcctggtgc taaagagggt accgttccaa cagacctaga
     1201 tcaagaaact ggtttagcta ggttagaatt attgggtaaa ttagagggta tcgatgtttt
     1261 cgacaccaaa ccattagatt cgtccaggaa gggtaccatg aaagatccga tcatcattga
     1321 atcttatgat gattatcgtt atgtcggttg tacgggttct cccgctggtt cacatactat
     1381 tatgtggtta aaaccaactg ttaacgaagt cgccagatgc tgggaatgtg gttctgttta
     1441 caaactaaac cctgttggtg ttccaaatga tgaccaccat cacctttctc ttgaagttgc
     1501 tgaagaaatg tctaaaggtg aagaattatt cactggtgtt gtcccaattt tggttgaatt
     1561 agatggtgat gttaatggtc acaaattttc tgtctccggt gaaggtgaag gtgatgctac
     1621 ttacggtaaa ttgaccttaa aatttatttg tactactggt aaattgccag ttccatggcc
     1681 aaccttagtc actactttaa cttatggtgt tcaatgtttt tctagatacc cagatcatat
     1741 gaaacaacat gactttttca agtctgccat gccagaaggt tatgttcaag aaagaactat
     1801 ttttttcaaa gatgacggta actacaagac cagagctgaa gtcaagtttg aaggtgatac
     1861 cttagttaat agaatcgaat taaaaggtat tgattttaaa gaagatggta acattttagg
     1921 tcacaaattg gaatacaact ataactctca caatgtttac atcatggctg acaaacaaaa
     1981 gaatggtatc aaagttaact tcaaaattag acacaacatt gaagatggtt ctgttcaatt
     2041 agctgaccat tatcaacaaa atactccaat tggtgatggt ccagtcttgt taccagacaa
     2101 ccattactta tccactcaat ctgccttatc caaagatcca aacgaaaaga gagaccacat
     2161 ggtcttgtta gaatttgtta ctgctgctgg tattacccat ggtatggatg aattgtacaa
     2221 agctgcaggt cgtgggattc ctgggttaat taacatcgct agcggaaagt cttacccaac
     2281 tgtgagtgct gattaccagg acgccgttga gaaggcgaag aagaagctca gaggcttcat
     2341 cgctgagaag agatgcgctc ctctaatgct ccgtttggca ttccactctg ctggaacctt
     2401 tgacaagggc acgaagaccg gtggaccctt cggaaccatc aagcaccctg ccgaactggc
     2461 tcacagcgct aacaacggtc ttgacatcgc tgttaggctt ttggagccac tcaaggcgga
     2521 gttccctatt ttgagctacg ccgatttcta ccagttggct ggcgttgttg ccgttgaggt
     2581 cacgggtgga cctaaggttc cattccaccc tggaagagag gacaagcctg agccaccacc
     2641 agagggtcgc ttgcccgatc ccactaaggg ttctgaccat ttgagagatg tgtttggcaa
     2701 agctatgggg cttactgacc aagatatcgt tgctctatct gggggtcaca ctattggagc
     2761 tgcacacaag gagcgttctg gatttgaggg tccctggacc tctaatcctc ttattttcga
     2821 caactcatac ttcacggagt tgttgagtgg tgagaaggaa ggtctccttc agctaccttc
     2881 tgacaaggct cttttgtctg accctgtatt ccgccctctc gttgacaaat atgcagcgga
     2941 cgaagatgcc ttctttgctg attacgctga ggctcaccaa aagctttccg agcttgggtt
     3001 tgctgatgcc ctgcagctgc ctcccctgga gcgcctgacc ctggacgctc agtgcagatc
     3061 ttagcctagg tgaggcgcgc cacttctaaa taagcgaatt tcttatgatt tatgattttt
     3121 attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt
     3181 ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag
     3241 gtatagtatg aggtcgctct tattgaccac acctctaccg gcagatccgc tagggataac
     3301 agggtaatat ctgcaggtcg acaaccctta atataacttc gtataatgta tgctatacga
     3361 agttattagg tctagagatc ccaatacaac agatcacgtg atctttttgt aagatgaagt
     3421 tgaagtagtg ttgcaccgtg ccaatgcagg tggctattag attaaatatg tgatttgttc
     3481 tattaagttt cctgtataat taatggggag cgctgattct cttttggtac gcttctcatc
     3541 cagcatttct gtatctttca ccttcaacct taggatctct acccttggcg aaaagtcctc
     3601 tgccaacaat gatgatatct gatccaccac ttacaacttc gtcgacggtt ctgtactgct
     3661 gacccaatgc atcgcctttg tcgtctaaac ctacacctgg ggtcatgatt agccaatcaa
     3721 acccttcttc tcttcctccc atatcgttct gagcaatgaa cccaataacg aaatctttat
     3781 cactctttgc aatatcaacg gtacccttag tatattcacc gtgtgctaga gaacccttgg
     3841 aagacaattc agcaagcatc aataatcccc ttggttcttt ggtgacctct tgcgcacctt
     3901 gtttcaagcc agcaacaata ccagcaccag taaccccgtg ggcgttggtg atatcagacc
     3961 attctgcgat acggtaaacg cccgatgtat attgtaattt gactgtgtta ccgatatcgg
     4021 cgaattttct gtcctcaaat atcaagaact tgtatttctc tgccaatgct ttcaatggaa
     4081 cgacagtacc ctcataactg aaatcatcca agatatcaac gtgtgttttc aaaaggcaaa
     4141 tgtatggacc caacgtttca acaagtttca atagctcatc agtcgaacga acgtcaagag
     4201 aagcacacaa attggtcttc ttttcatcca ttaaacgtaa aagtttcgat gcaaccggac
     4261 ttgcatgagt ctcagctcta ctggtatatg attttgtgga catggtgcaa ctaattgacg
     4321 ggagtgtatt gacgctggcg tactggcttt cacaaaatgg cccaatcaca accacatctt
     4381 agatagttga aatgacttta gataacatca attgagatga gcttaatcat gtcaaagcta
     4441 aaagtgtcac catgaacgac aattcttaag caaatcacgt gatatagatc cacgaataac
     4501 caccatttga tgctcgaggc aagtaatgtg tgtaaaaaaa tgcgttacca ccatccaatg
     4561 cagaccgatc ttctacccag aatcacatat atttatgtac cgagtacctt ttttctatct
     4621 tccaattgct tctcccatat gattgtctcc gtaagctcga aatttctaag ttggatttta
     4681 atcttcacgc aggatgacag ttcgatgagc ttctgaggag tgtttagaac ataatcattt
     4741 atccatggtc tatctcttct tgtcgctttt tctcctcgat agaacctaaa taaaacgagc
     4801 tctcgagaac ccttaatata acttcgtata atgtatgcta tacgaagtta ttaggtgata
     4861 tcagatccac tagtggccta tgcggccgcg gatctgccgg tctccctata gtgagtcgta
     4921 ttaatttcga taagccaggt taacctgcat taatgaatcg gccaacgcgc ggggagaggc
     4981 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5041 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5101 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5161 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5221 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5281 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5341 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5401 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5461 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5521 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5581 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5641 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5701 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5761 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5821 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5881 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5941 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6001 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6061 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6121 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6181 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6241 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6301 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6361 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6421 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6481 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6541 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6601 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6661 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6721 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6781 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6841 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6901 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     6961 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     7021 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     7081 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     7141 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgga catattgtcg
     7201 ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg atttaggtga
     7261 cactata
//
