LOCUS       sequence-424257-        7580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing Pex3-4V5-APEX2. Insufficient signal for EM. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7580)
  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 7580)
  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-Pex3-4V5-APEX2-URA
FEATURES             Location/Qualifiers
     source          1..7580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2394..2435
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2442..2483
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2490..2531
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2538..2579
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2586..3332
                     /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             3339..3365
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      3407..3594
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3462..3486)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    3646..3679
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5131..5164
                     /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(5217..5236)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5218..5236)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5323..5340)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5494..6082)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5574..5593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6253..7044,7045..7113))
                     /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     6876..6895
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7114..7218)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7286..7304
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7342..7364)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7464..7483
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7564..7580,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7564..7580,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg tgggattcct ggtgttaatc
       61 attatccatt tttgctttgc ccttgccttt tgctttgcag cgtctaagtc gagtttggac
      121 atgattgaaa atggaggcct aaataacaaa gctaatttct gctccttgga aatggtttta
      181 ggctttctca ctaatttggt gtactccttg atattgccgt cttcatcaag ttcgatgacc
      241 ggctcttcca catactcata ttcgtcgtcg tcattttctt cctcggagtc caaggaatct
      301 gaagagttgt cattgatgta tgtattagta tttgtgtcat ccaatcgttg attaaaaatg
      361 ctttcaggtc ttccttcgtc aaacatgtcg ttcaccctat gcaatggttc aaatcttccg
      421 ccaatgccgc cgtaagaaat tgggaatcct gaagtttcag taagtacttc atgtcttgcc
      481 tcatcaggtg gcctgacata atcttggtca ggctgttgtt gttggtaagc ttccctttgc
      541 aacctctccg ctaattgtgc atcggcatct gaatctacag aggtgggcgc cataggagtc
      601 gaattagacg gagtaggtgt gttgttactg ctcagtaaag cagccccacc attttcgaaa
      661 aacaatgata tggctgtgtt caagttcccg tctgccattt cgatgaattg agtggcaata
      721 gcagcgtcat cagcgccagt gatggccata aattcgtcta cttttccctc tgacatataa
      781 aaggtatgtt attattgcca cacgtcgtag tctatgcgtt tgtagtgaca ctattgagcg
      841 atcatagcta agatgtcgtt aacaagtagt agagtttgcg tgaatgtatg aatatttccc
      901 tttttcctta aaaattcctt gcattttggt aatcgtgtgt aatgataagg gcgttattcg
      961 acttatatga gcatggggta tattattgag gtagttaata ctagtcatcg taaaagcaga
     1021 agcacgaaac aaggaggcaa accactaaaa ggatggcccc aaatcaaaga tcacgttcgc
     1081 ttctgcagag acatcgagga aaggtactca tttcattgac tggaatagct gctttattta
     1141 ccacagggtc ggtggtagtg tttttcgtga agagatggtt gtataaacag cagttacgga
     1201 ttactgagca acacttcatc aaagaacaga tcaaaagaag gttcgagcag acacaggaag
     1261 actcgttgta cacaatatac gagctacttc ctgtatggag aatggttttg aatgaaaacg
     1321 atttgaattt ggacagcatc gttacccaat taaaggacca aaagaaccag ttgactagag
     1381 caaagtctag tgaaagcaga gaaagttcgc cgttaaaaag taaagctgag ttgtggaacg
     1441 agttagaact aaagagtttg atcaagctgg tgacagtgac gtacacagta tcgtcgctga
     1501 ttcttttaac aagactgcaa ctaaatatcc tgacaagaaa cgagtacctg gactccgcaa
     1561 taaaattaac catgcagcag gaaaactgca acaaactaca gaataggttc tataactggg
     1621 ttacatcgtg gtggagcgat ccggaggaca aagcggatga tgcaatggta atggcagcaa
     1681 aaaagtcaaa gaaagaaggc caggaagtgt atattaatga gcaagctttc ctatcactat
     1741 catggtggat tcttaacaag gggtggttga gttacaatga aataattaca aatcaaatcg
     1801 aaattgaatt tgacggcata cacccaagag atactctaac gttagaagaa tttagttctc
     1861 gtctcacgaa catattccgg aacacgaatt cccaaatatt ccagcaaaat aataataacc
     1921 tcacgtctat tttgcttcct aaagattcca gtggacagga gtttcttctg tcacagacgc
     1981 ttgacgcgga tgctttaaca agttttcact ctaacacact agtttttaat caattggtga
     2041 atgaactgac ccaatgtatc gaaagcacag caacatccat agtgttagag tctctaataa
     2101 atgaatcatt ccatttcata atgaacaagg tcggcataaa gacaatagcc aagaaaaaac
     2161 ctggccaaga agatcagcaa cagtatcaaa tggcagtttt cgctatgtcg atgaaagact
     2221 gttgccaaga aatgcttcaa acaactgccg ggtcttccca tagcggcagc gtgaacgaat
     2281 acctggccac tctggattct gtgcagccgc tcgatgatct gagcgccagc gtatacagca
     2341 actttggcgt ctccagctcg ttttccttca agcctttaat taacatcgct agcggtaaac
     2401 caattccaaa tccattgttg ggtttggatt ctactcctag cggtaaacca attccaaatc
     2461 cattgttggg tttggattct actcctagcg gtaaaccaat tccaaatcca ttgttgggtt
     2521 tggattctac tcctagcggt aaaccaattc caaatccatt gttgggtttg gattctactc
     2581 ctaggggaaa gtcttaccca actgtgagtg ctgattacca ggacgccgtt gagaaggcga
     2641 agaagaagct cagaggcttc atcgctgaga agagatgcgc tcctctaatg ctccgtttgg
     2701 cattccactc tgctggaacc tttgacaagg gcacgaagac cggtggaccc ttcggaacca
     2761 tcaagcaccc tgccgaactg gctcacagcg ctaacaacgg tcttgacatc gctgttaggc
     2821 ttttggagcc actcaaggcg gagttcccta ttttgagcta cgccgatttc taccagttgg
     2881 ctggcgttgt tgccgttgag gtcacgggtg gacctaaggt tccattccac cctggaagag
     2941 aggacaagcc tgagccacca ccagagggtc gcttgcccga tcccactaag ggttctgacc
     3001 atttgagaga tgtgtttggc aaagctatgg ggcttactga ccaagatatc gttgctctat
     3061 ctgggggtca cactattgga gctgcacaca aggagcgttc tggatttgag ggtccctgga
     3121 cctctaatcc tcttattttc gacaactcat acttcacgga gttgttgagt ggtgagaagg
     3181 aaggtctcct tcagctacct tctgacaagg ctcttttgtc tgaccctgta ttccgccctc
     3241 tcgttgacaa atatgcagcg gacgaagatg ccttctttgc tgattacgct gaggctcacc
     3301 aaaagctttc cgagcttggg tttgctgatg ccctgcagct gcctcccctg gagcgcctga
     3361 ccctggacgc tcagtgcaga tcttgaggcg cgccacttct aaataagcga atttcttatg
     3421 atttatgatt tttattatta aataagttat aaaaaaaata agtgtataca aattttaaag
     3481 tgactcttag gttttaaaac gaaaattctt attcttgagt aactctttcc tgtaggtcag
     3541 gttgctttct caggtatagt atgaggtcgc tcttattgac cacacctcta ccggcagatc
     3601 cgctagggat aacagggtaa tatctgcagg tcgacaaccc ttaatataac ttcgtataat
     3661 gtatgctata cgaagttatt aggtctagag atcccaatac aacagatcac gtgatctttt
     3721 tgtaagatga agttgaagta gtgttgcacc gtgccaatgc aggtggctat tagattaaat
     3781 atgtgatttg ttctattaag tttcctgtat aattaatggg gagcgctgat tctcttttgg
     3841 tacgcttccc atccagcatt tctgtatctt tcaccttcaa ccttaggatc tctacccttg
     3901 gcgaaaagtc ctctgccaac aatgatgata tctgatccac cacttacaac ttcgtcgacg
     3961 gttctgtact gctgacccaa tgcatcgcct ttgtcgtcta aacctacacc tggggtcatg
     4021 attagccaat caaacccttc ttctcttcct cccatatcgt tctgagcaat gaacccaata
     4081 acgaaatctt tatcactctt tgcaatatca acggtaccct tagtatattc accgtgtgct
     4141 agagaaccct tggaagacaa ttcagcaagc atcaataatc cccttggttc tttggtgacc
     4201 tcttgcgcac cttgtttcaa gccagcaaca ataccagcac cagtaacccc gtgggcgttg
     4261 gtgatatcag accattctgc gatacggtaa acgcccgatg tatattgtaa tttgactgtg
     4321 ttaccgatat cggcgaattt tctgtcctca aatatcaaga acttgtattt ctctgccaat
     4381 gctttcaatg gaacgacagt accctcataa ctgaaatcat ccaagatatc aacgtgtgtt
     4441 ttcaaaaggc aaatgtatgg acccaacgtt tcaacaagtt tcaatagctc atcagtcgaa
     4501 cgaacgtcaa gagaagcaca caaattggtc ttcttttcat ccattaaacg taaaagtttc
     4561 gatgcaaccg gacttgcatg agtctcagct ctactggtat atgattttgt ggacatggtg
     4621 caactaattg acgggagtgt attgacgctg gcgtactggc tttcacaaaa tggcccaatc
     4681 acaaccacat cttagatagt tgaaatgact ttagataaca tcaattgaga tgagcttaat
     4741 catgtcaaag ctaaaagtgt caccatgaac gacaattctt aagcaaatca cgtgatatag
     4801 atccacgaat aaccaccatt tgatgctcga ggcaagtaat gtgtgtaaaa aaatgcgtta
     4861 ccaccatcca atgcagaccg atcttctacc cagaatcaca tatatttatg taccgagtac
     4921 cttttttcta tcttccaatt gcttctccca tatgattgtc tccgtaagct cgaaatttct
     4981 aagttggatt ttaatcttca cgcaggatga cagttcgatg agcttctgag gagtgtttag
     5041 aacataatca tttatccatg gtctatctct tcttgtcgct ttttctcctc gatagaacct
     5101 aaataaaacg agctctcgag aacccttaat ataacttcgt ataatgtatg ctatacgaag
     5161 ttattaggtg atatcagatc cactagtggc ctatgcggcc gcggatctgc cggtctccct
     5221 atagtgagtc gtattaattt cgataagcca ggttaacctg cattaatgaa tcggccaacg
     5281 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5341 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5401 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5461 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5521 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5581 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5641 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5701 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5761 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5821 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5881 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5941 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6001 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6061 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6121 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6181 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6241 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6301 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6361 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6421 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6481 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6541 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6601 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6661 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6721 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6781 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6841 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6901 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6961 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7021 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7081 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7141 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7201 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     7261 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     7321 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     7381 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     7441 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     7501 ggacatattg tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat
     7561 acgatttagg tgacactata
//
