LOCUS       sequence-424587-        6536 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing Cox4-V5-APEX2. Marker for mitochondrial inner membrane. 
            Tends to generate facture in thin resin sections..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6536)
  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 6536)
  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-V5-APEX2-Ura
FEATURES             Location/Qualifiers
     source          1..6536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1026..1091
                     /codon_start=1
                     /product="mitochondrial presequence of yeast cytochrome c 
                     oxidase subunit IV (Hurt et al., 1984)"
                     /label=pre-CoxIV
                     /translation="MLSLRQSIRFFKPATRTLCSSR"
     CDS             1491..1532
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1533..2279
                     /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             2286..2312
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      2363..2550
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2418..2442)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    2602..2635
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4087..4120
                     /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(4173..4192)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4174..4192)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4279..4296)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4450..5038)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4530..4549)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5209..6000,6001..6069))
                     /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     5832..5851
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6070..6174)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6242..6260
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6298..6320)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6420..6439
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6520..6536,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6520..6536,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgccta ttggattggc ccaattgctg ccaaatttgg
       61 tgaatatggt ggtgatgtcg caatgtggtt aagtatggcg ttctcaggtg ttgtataccc
      121 tccttgtaga tacttagagc tacgtaaatt tggtcgttaa ttgaaatgac ataagtatat
      181 attaaaacta cggtgcagcg gcaaacttta gtttagataa tattggcagg tagattcatg
      241 gtatgttaaa ttacgctcag ggaggatatt tttttgtaca cattatcttt tcacatgaaa
      301 atctaagcct ggtggacaat tgaaatcatc gagtgatata ctgacacttt gaacagtagc
      361 acaaatcgtt taatttgctg tcacgatcta agttttctgt cagctgactg tccaccaata
      421 agatcagcgg attccaaagg cgtccttcga aaaagcgata tctcccatct tggttagtag
      481 caaataaagg gaagagaaaa taagacacac tcaaggataa aatacttcgc ttcttccaag
      541 tataaacaca ggaaagaaag atatacgaag attagaattt ttttcatact ttttacaaaa
      601 catttttttg tacttaggca cgctaattgc gtaaaaatta gcataaacag gaaataagat
      661 tattttgaaa tgtatgttat aatcctctta ttgaaaaata catgagatgt aggagaagaa
      721 ctaccaggaa agaagatatt taacagatcg aacgaaacgg aaaagaacta agagaaaaca
      781 aatctggatt tgactttaag aaaaaattta tttgcaagtt taactcgttc cctgcttttc
      841 tatttggctt gagacgataa ttgcataatt gtccaaattg tccatcatat aatgaagaat
      901 tcgatgcgaa atattccctg tcactttcct ttagtcaacg tattcttctc cctgaagaaa
      961 cagtatacta acaatactca cccatttcga ttttgatgtt gccatacaaa tagataacaa
     1021 gcacaatgct ttcactacgt caatctataa gatttttcaa gccagccaca agaactttgt
     1081 gtagctctag atatctgctt cagcaaaaac ccgtggtgaa aactgcccaa aacttagcag
     1141 aagttaatgg tccagaaact ttgattggtc ctggtgctaa agagggtacc gttccaacag
     1201 acctagatca agaaactggt ttagctaggt tagaattatt gggtaaatta gagggtatcg
     1261 atgttttcga caccaaacca ttagattcgt ccaggaaggg taccatgaaa gatccgatca
     1321 tcattgaatc ttatgatgat tatcgttatg tcggttgtac gggttctccc gctggttcac
     1381 atactattat gtggttaaaa ccaactgtta acgaagtcgc cagatgctgg gaatgtggtt
     1441 ctgtttacaa actaaaccct gttggtgttc caaatgatga ccaccatcac ggtaaaccaa
     1501 ttccaaatcc attgttgggt ttggattcta ctggaaagtc ttacccaact gtgagtgctg
     1561 attaccagga cgccgttgag aaggcgaaga agaagctcag aggcttcatc gctgagaaga
     1621 gatgcgctcc tctaatgctc cgtttggcat tccactctgc tggaaccttt gacaagggca
     1681 cgaagaccgg tggacccttc ggaaccatca agcaccctgc cgaactggct cacagcgcta
     1741 acaacggtct tgacatcgct gttaggcttt tggagccact caaggcggag ttccctattt
     1801 tgagctacgc cgatttctac cagttggctg gcgttgttgc cgttgaggtc acgggtggac
     1861 ctaaggttcc attccaccct ggaagagagg acaagcctga gccaccacca gagggtcgct
     1921 tgcccgatcc cactaagggt tctgaccatt tgagagatgt gtttggcaaa gctatggggc
     1981 ttactgacca agatatcgtt gctctatctg ggggtcacac tattggagct gcacacaagg
     2041 agcgttctgg atttgagggt ccctggacct ctaatcctct tattttcgac aactcatact
     2101 tcacggagtt gttgagtggt gagaaggaag gtctccttca gctaccttct gacaaggctc
     2161 ttttgtctga ccctgtattc cgccctctcg ttgacaaata tgcagcggac gaagatgcct
     2221 tctttgctga ttacgctgag gctcaccaaa agctttccga gcttgggttt gctgatgccc
     2281 tgcagctgcc tcccctggag cgcctgaccc tggacgctca gtgcagatct tagcctaggt
     2341 gaggcgcgcc acttctaaat aagcgaattt cttatgattt atgattttta ttattaaata
     2401 agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa
     2461 attcttattc ttgagtaact ctttcctgta ggtcaggttg ctttctcagg tatagtatga
     2521 ggtcgctctt attgaccaca cctctaccgg cagatccgct agggataaca gggtaatatc
     2581 tgcaggtcga caacccttaa tataacttcg tataatgtat gctatacgaa gttattaggt
     2641 ctagagatcc caatacaaca gatcacgtga tctttttgta agatgaagtt gaagtagtgt
     2701 tgcaccgtgc caatgcaggt ggctattaga ttaaatatgt gatttgttct attaagtttc
     2761 ctgtataatt aatggggagc gctgattctc ttttggtacg cttcccatcc agcatttctg
     2821 tatctttcac cttcaacctt aggatctcta cccttggcga aaagtcctct gccaacaatg
     2881 atgatatctg atccaccact tacaacttcg tcgacggttc tgtactgctg acccaatgca
     2941 tcgcctttgt cgtctaaacc tacacctggg gtcatgatta gccaatcaaa cccttcttct
     3001 cttcctccca tatcgttctg agcaatgaac ccaataacga aatctttatc actctttgca
     3061 atatcaacgg tacccttagt atattcaccg tgtgctagag aacccttgga agacaattca
     3121 gcaagcatca ataatcccct tggttctttg gtgacctctt gcgcaccttg tttcaagcca
     3181 gcaacaatac cagcaccagt aaccccgtgg gcgttggtga tatcagacca ttctgcgata
     3241 cggtaaacgc ccgatgtata ttgtaatttg actgtgttac cgatatcggc gaattttctg
     3301 tcctcaaata tcaagaactt gtatttctct gccaatgctt tcaatggaac gacagtaccc
     3361 tcataactga aatcatccaa gatatcaacg tgtgttttca aaaggcaaat gtatggaccc
     3421 aacgtttcaa caagtttcaa tagctcatca gtcgaacgaa cgtcaagaga agcacacaaa
     3481 ttggtcttct tttcatccat taaacgtaaa agtttcgatg caaccggact tgcatgagtc
     3541 tcagctctac tggtatatga ttttgtggac atggtgcaac taattgacgg gagtgtattg
     3601 acgctggcgt actggctttc acaaaatggc ccaatcacaa ccacatctta gatagttgaa
     3661 atgactttag ataacatcaa ttgagatgag cttaatcatg tcaaagctaa aagtgtcacc
     3721 atgaacgaca attcttaagc aaatcacgtg atatagatcc acgaataacc accatttgat
     3781 gctcgaggca agtaatgtgt gtaaaaaaat gcgttaccac catccaatgc agaccgatct
     3841 tctacccaga atcacatata tttatgtacc gagtaccttt tttctatctt ccaattgctt
     3901 ctcccatatg attgtctccg taagctcgaa atttctaagt tggattttaa tcttcacgca
     3961 ggatgacagt tcgatgagct tctgaggagt gtttagaaca taatcattta tccatggtct
     4021 atctcttctt gtcgcttttt ctcctcgata gaacctaaat aaaacgagct ctcgagaacc
     4081 cttaatataa cttcgtataa tgtatgctat acgaagttat taggtgatat cagatccact
     4141 agtggcctat gcggccgcgg atctgccggt ctccctatag tgagtcgtat taatttcgat
     4201 aagccaggtt aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4261 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4321 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4381 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4441 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4501 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4561 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4621 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4681 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4741 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4801 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4861 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4921 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4981 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5041 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5101 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5161 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5221 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5281 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5341 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5401 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5461 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5521 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5581 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5641 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5701 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5761 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5821 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5881 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5941 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6001 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6061 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6121 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6181 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     6241 aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct
     6301 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     6361 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc
     6421 ggcatcagag cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg
     6481 ctacaattaa tacataacct tatgtatcat acacatacga tttaggtgac actata
//
