LOCUS       sequence-422536-        6270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing OM14-V5-APEX2. Marker for mitochondrial outer membrane. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6270)
  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 6270)
  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-OM14-V5-APEX2-Ura
FEATURES             Location/Qualifiers
     source          1..6270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1225..1266
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1267..2013
                     /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             2020..2046
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      2097..2284
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2152..2176)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    2336..2369
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3821..3854
                     /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(3907..3926)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3908..3926)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4013..4030)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4184..4772)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4264..4283)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4943..5734,5735..5803))
                     /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     5566..5585
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5804..5908)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5976..5994
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6032..6054)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6154..6173
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6254..6270,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6254..6270,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttctggag tcgttcttga cttttacttt ggtcgcttga
       61 tgtttctctc gtcttcccgt tcgctctttt atttttttat tttttttttc ctttgacttc
      121 gcatgaatga gttcattcat gaagcgaaag gagagattct atataccacg caaccaaata
      181 tgtaagtata taaaatatat gtatgtgtgt gcactaattg gcggcatact ctttcttgac
      241 ccgttgtgat agggccttag ctgcttccca ttgcctgtcc cgagtccagc gactcaagac
      301 gttggacgtg cgcacgtact cacacatggc cgaattggcc cttacgaatg agctcttggc
      361 tacccctctt gctgcactcg ccacacacca taactggtat aattcgtttc tcatgctagt
      421 ctcttttttt ttttttactt gtttcctttt cctttgttgc ttatccgctt tctcgttctc
      481 tcctctgttc ttgcctcaac tcctcaaatc tctgctgtcc cacttacgtt ttattttttc
      541 accgtcccct ttccctgccc tactaccccg ggccagcggc caatgacgcc gccgctacct
      601 cctctcccgt taatcgtccc tgcgtatata aagtgtcgat caatgtgccc acttcaaccg
      661 aaaccgccga cgtaacccta ttccaacaat tagaaacata acatctacct ccagcatctc
      721 ataatatgtc tgcaacgtat gtacactaat atctgaaacg tatcgcctaa tcttttctcc
      781 cggttctctt ttgactaacg tatatcatct atgaccatct ttcttttccc cagtgctaaa
      841 cacgacagta acgctagccc aaattctgac tctgaagacg gtcaccacca caacaataag
      901 aaggaatgcg ctatcgaata tctgaaggca cggttaaata gtgcatcagc agtcgcttgc
      961 ggctacctcc aagcttttgt cagtaagacg caagactttg ccaaagtatg ctttttagaa
     1021 cttcagaatc ccgttgtctt ggtcaacttg ttgctgcatt cttcagtggt atgttattta
     1081 tgtaacgggt atgcgaacca caacgccaga ttcttgaagg ggaaacctaa ctctacagtc
     1141 ttagcaacaa ccgccggcgc tctgggtctt ttgacgctgg acggtataat ttcaaagaaa
     1201 tactactcca gatacgacaa gaaaggtaaa ccaattccaa atccattgtt gggtttggat
     1261 tctactggaa agtcttaccc aactgtgagt gctgattacc aggacgccgt tgagaaggcg
     1321 aagaagaagc tcagaggctt catcgctgag aagagatgcg ctcctctaat gctccgtttg
     1381 gcattccact ctgctggaac ctttgacaag ggcacgaaga ccggtggacc cttcggaacc
     1441 atcaagcacc ctgccgaact ggctcacagc gctaacaacg gtcttgacat cgctgttagg
     1501 cttttggagc cactcaaggc ggagttccct attttgagct acgccgattt ctaccagttg
     1561 gctggcgttg ttgccgttga ggtcacgggt ggacctaagg ttccattcca ccctggaaga
     1621 gaggacaagc ctgagccacc accagagggt cgcttgcccg atcccactaa gggttctgac
     1681 catttgagag atgtgtttgg caaagctatg gggcttactg accaagatat cgttgctcta
     1741 tctgggggtc acactattgg agctgcacac aaggagcgtt ctggatttga gggtccctgg
     1801 acctctaatc ctcttatttt cgacaactca tacttcacgg agttgttgag tggtgagaag
     1861 gaaggtctcc ttcagctacc ttctgacaag gctcttttgt ctgaccctgt attccgccct
     1921 ctcgttgaca aatatgcagc ggacgaagat gccttctttg ctgattacgc tgaggctcac
     1981 caaaagcttt ccgagcttgg gtttgctgat gccctgcagc tgcctcccct ggagcgcctg
     2041 accctggacg ctcagtgcag atcttagcct aggtgaggcg cgccacttct aaataagcga
     2101 atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata agtgtataca
     2161 aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt aactctttcc
     2221 tgtaggtcag gttgctttct caggtatagt atgaggtcgc tcttattgac cacacctcta
     2281 ccggcagatc cgctagggat aacagggtaa tatctgcagg tcgacaaccc ttaatataac
     2341 ttcgtataat gtatgctata cgaagttatt aggtctagag atcccaatac aacagatcac
     2401 gtgatctttt tgtaagatga agttgaagta gtgttgcacc gtgccaatgc aggtggctat
     2461 tagattaaat atgtgatttg ttctattaag tttcctgtat aattaatggg gagcgctgat
     2521 tctcttttgg tacgcttccc atccagcatt tctgtatctt tcaccttcaa ccttaggatc
     2581 tctacccttg gcgaaaagtc ctctgccaac aatgatgata tctgatccac cacttacaac
     2641 ttcgtcgacg gttctgtact gctgacccaa tgcatcgcct ttgtcgtcta aacctacacc
     2701 tggggtcatg attagccaat caaacccttc ttctcttcct cccatatcgt tctgagcaat
     2761 gaacccaata acgaaatctt tatcactctt tgcaatatca acggtaccct tagtatattc
     2821 accgtgtgct agagaaccct tggaagacaa ttcagcaagc atcaataatc cccttggttc
     2881 tttggtgacc tcttgcgcac cttgtttcaa gccagcaaca ataccagcac cagtaacccc
     2941 gtgggcgttg gtgatatcag accattctgc gatacggtaa acgcccgatg tatattgtaa
     3001 tttgactgtg ttaccgatat cggcgaattt tctgtcctca aatatcaaga acttgtattt
     3061 ctctgccaat gctttcaatg gaacgacagt accctcataa ctgaaatcat ccaagatatc
     3121 aacgtgtgtt ttcaaaaggc aaatgtatgg acccaacgtt tcaacaagtt tcaatagctc
     3181 atcagtcgaa cgaacgtcaa gagaagcaca caaattggtc ttcttttcat ccattaaacg
     3241 taaaagtttc gatgcaaccg gacttgcatg agtctcagct ctactggtat atgattttgt
     3301 ggacatggtg caactaattg acgggagtgt attgacgctg gcgtactggc tttcacaaaa
     3361 tggcccaatc acaaccacat cttagatagt tgaaatgact ttagataaca tcaattgaga
     3421 tgagcttaat catgtcaaag ctaaaagtgt caccatgaac gacaattctt aagcaaatca
     3481 cgtgatatag atccacgaat aaccaccatt tgatgctcga ggcaagtaat gtgtgtaaaa
     3541 aaatgcgtta ccaccatcca atgcagaccg atcttctacc cagaatcaca tatatttatg
     3601 taccgagtac cttttttcta tcttccaatt gcttctccca tatgattgtc tccgtaagct
     3661 cgaaatttct aagttggatt ttaatcttca cgcaggatga cagttcgatg agcttctgag
     3721 gagtgtttag aacataatca tttatccatg gtctatctct tcttgtcgct ttttctcctc
     3781 gatagaacct aaataaaacg agctctcgag aacccttaat ataacttcgt ataatgtatg
     3841 ctatacgaag ttattaggtg atatcagatc cactagtggc ctatgcggcc gcggatctgc
     3901 cggtctccct atagtgagtc gtattaattt cgataagcca ggttaacctg cattaatgaa
     3961 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4021 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4081 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4141 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4201 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4261 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4321 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4381 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4441 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4501 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4561 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4621 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4681 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4741 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4801 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4861 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4921 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4981 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5041 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5101 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5161 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5221 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5281 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5341 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5401 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5461 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5521 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5581 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5641 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5701 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5761 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5821 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5881 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     5941 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     6001 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     6061 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     6121 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
     6181 tgcaccatat ggacatattg tcgttagaac gcggctacaa ttaatacata accttatgta
     6241 tcatacacat acgatttagg tgacactata
//
