LOCUS       sequence-424567-        7546 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Moderate overexpression of Pex3-4V5-APEX2 under LSP1 promoter. 
            Marker for peroxisomes. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7546)
  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 7546)
  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-pLSP1-Pex3-4V5-APEX2-URA
FEATURES             Location/Qualifiers
     source          1..7546
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2360..2401
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2408..2449
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2456..2497
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2504..2545
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2552..3298
                     /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             3305..3331
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      3373..3560
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3428..3452)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    3612..3645
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5097..5130
                     /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(5183..5202)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5184..5202)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5289..5306)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5460..6048)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5540..5559)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6219..7010,7011..7079))
                     /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     6842..6861
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7080..7184)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7252..7270
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7308..7330)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7430..7449
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7530..7546,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7530..7546,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttttacca tccctttcaa gttttgcaat acacttcatg
       61 agcaaaacag tatatggaat accagaaaac gtggctgtat ccattttact taggtcaaat
      121 gtatctacgt aatttattag ttcaggccaa gggttctgta accgtaaatc atacttctta
      181 ctatctgggt gtgcttgcaa aacaaaatgt gtttctgaga gtaccagata tatatatcca
      241 taaaatccag agacgaaagt gtttagaaca ggcggaaact ttgttcctct ctgccgccta
      301 atctcattta aatcgagcat cgcagtttgc tcaccaatac aagtcaagat caccaccgag
      361 aagcgggtcc aatcatactg aggttgctgt aacgtctttc taaggtcatt ttcctcatat
      421 tctagctgtt tagaagccaa tggctcaaga tcttttttaa gctctgctaa aaacagagaa
      481 cccgattgaa ctgcacactc tactttgagc caagtaagag atgatattcc ggcaagcacc
      541 agattcttaa aaacttcctg cagcaaaggt gtagcaggtc ctaccacgca cacacgggac
      601 cggttcaaac tatcttggcc caatgctccc catagtctca gctgtctatc atatctttcc
      661 atattttcat ctacggctgt ctagttggcc taagaaagaa agagaggcga ctcatttttt
      721 tgacgtcaat cgtctgactt gtccttctat ttcattgcta ggctagaaag cttagctctt
      781 acaaccccgc tcgttgcagc cgagtgaaaa ggggattgcg aaaggcgtta gaggggataa
      841 aaaggggccc tttaaaaaaa agacggaaaa accgggaaga tgcaacggct aaatcgcaat
      901 atataaacag gtgagataga aaagaaaaaa atcagttaac ctctttcctc aattccaatg
      961 cttgaccgta aattctcttt atttggtata cgaaagatta aactttcaaa gctccaacac
     1021 aagacgtacg atggccccaa atcaaagatc acgttcgctt ctgcagagac atcgaggaaa
     1081 ggtactcatt tcattgactg gaatagctgc tttatttacc acagggtcgg tggtagtgtt
     1141 tttcgtgaag agatggttgt ataaacagca gttacggatt actgagcaac acttcatcaa
     1201 agaacagatc aaaagaaggt tcgagcagac acaggaagac tcgttgtaca caatatacga
     1261 gctacttcct gtatggagaa tggttttgaa tgaaaacgat ttgaatttgg acagcatcgt
     1321 tacccaatta aaggaccaaa agaaccagtt gactagagca aagtctagtg aaagcagaga
     1381 aagttcgccg ttaaaaagta aagctgagtt gtggaacgag ttagaactaa agagtttgat
     1441 caagctggtg acagtgacgt acacagtatc gtcgctgatt cttttaacaa gactgcaact
     1501 aaatatcctg acaagaaacg agtacctgga ctccgcaata aaattaacca tgcagcagga
     1561 aaactgcaac aaactacaga ataggttcta taactgggtt acatcgtggt ggagcgatcc
     1621 ggaggacaaa gcggatgatg caatggtaat ggcagcaaaa aagtcaaaga aagaaggcca
     1681 ggaagtgtat attaatgagc aagctttcct atcactatca tggtggattc ttaacaaggg
     1741 gtggttgagt tacaatgaaa taattacaaa tcaaatcgaa attgaatttg acggcataca
     1801 cccaagagat actctaacgt tagaagaatt tagttctcgt ctcacgaaca tattccggaa
     1861 cacgaattcc caaatattcc agcaaaataa taataacctc acgtctattt tgcttcctaa
     1921 agattccagt ggacaggagt ttcttctgtc acagacgctt gacgcggatg ctttaacaag
     1981 ttttcactct aacacactag tttttaatca attggtgaat gaactgaccc aatgtatcga
     2041 aagcacagca acatccatag tgttagagtc tctaataaat gaatcattcc atttcataat
     2101 gaacaaggtc ggcataaaga caatagccaa gaaaaaacct ggccaagaag atcagcaaca
     2161 gtatcaaatg gcagttttcg ctatgtcgat gaaagactgt tgccaagaaa tgcttcaaac
     2221 aactgccggg tcttcccata gcggcagcgt gaacgaatac ctggccactc tggattctgt
     2281 gcagccgctc gatgatctga gcgccagcgt atacagcaac tttggcgtct ccagctcgtt
     2341 ttccttcaag cctgctagcg gtaaaccaat tccaaatcca ttgttgggtt tggattctac
     2401 tcctagcggt aaaccaattc caaatccatt gttgggtttg gattctactc ctagcggtaa
     2461 accaattcca aatccattgt tgggtttgga ttctactcct agcggtaaac caattccaaa
     2521 tccattgttg ggtttggatt ctactcctag gggaaagtct tacccaactg tgagtgctga
     2581 ttaccaggac gccgttgaga aggcgaagaa gaagctcaga ggcttcatcg ctgagaagag
     2641 atgcgctcct ctaatgctcc gtttggcatt ccactctgct ggaacctttg acaagggcac
     2701 gaagaccggt ggacccttcg gaaccatcaa gcaccctgcc gaactggctc acagcgctaa
     2761 caacggtctt gacatcgctg ttaggctttt ggagccactc aaggcggagt tccctatttt
     2821 gagctacgcc gatttctacc agttggctgg cgttgttgcc gttgaggtca cgggtggacc
     2881 taaggttcca ttccaccctg gaagagagga caagcctgag ccaccaccag agggtcgctt
     2941 gcccgatccc actaagggtt ctgaccattt gagagatgtg tttggcaaag ctatggggct
     3001 tactgaccaa gatatcgttg ctctatctgg gggtcacact attggagctg cacacaagga
     3061 gcgttctgga tttgagggtc cctggacctc taatcctctt attttcgaca actcatactt
     3121 cacggagttg ttgagtggtg agaaggaagg tctccttcag ctaccttctg acaaggctct
     3181 tttgtctgac cctgtattcc gccctctcgt tgacaaatat gcagcggacg aagatgcctt
     3241 ctttgctgat tacgctgagg ctcaccaaaa gctttccgag cttgggtttg ctgatgccct
     3301 gcagctgcct cccctggagc gcctgaccct ggacgctcag tgcagatctt gaggcgcgcc
     3361 acttctaaat aagcgaattt cttatgattt atgattttta ttattaaata agttataaaa
     3421 aaaataagtg tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa attcttattc
     3481 ttgagtaact ctttcctgta ggtcaggttg ctttctcagg tatagtatga ggtcgctctt
     3541 attgaccaca cctctaccgg cagatccgct agggataaca gggtaatatc tgcaggtcga
     3601 caacccttaa tataacttcg tataatgtat gctatacgaa gttattaggt ctagagatcc
     3661 caatacaaca gatcacgtga tctttttgta agatgaagtt gaagtagtgt tgcaccgtgc
     3721 caatgcaggt ggctattaga ttaaatatgt gatttgttct attaagtttc ctgtataatt
     3781 aatggggagc gctgattctc ttttggtacg cttcccatcc agcatttctg tatctttcac
     3841 cttcaacctt aggatctcta cccttggcga aaagtcctct gccaacaatg atgatatctg
     3901 atccaccact tacaacttcg tcgacggttc tgtactgctg acccaatgca tcgcctttgt
     3961 cgtctaaacc tacacctggg gtcatgatta gccaatcaaa cccttcttct cttcctccca
     4021 tatcgttctg agcaatgaac ccaataacga aatctttatc actctttgca atatcaacgg
     4081 tacccttagt atattcaccg tgtgctagag aacccttgga agacaattca gcaagcatca
     4141 ataatcccct tggttctttg gtgacctctt gcgcaccttg tttcaagcca gcaacaatac
     4201 cagcaccagt aaccccgtgg gcgttggtga tatcagacca ttctgcgata cggtaaacgc
     4261 ccgatgtata ttgtaatttg actgtgttac cgatatcggc gaattttctg tcctcaaata
     4321 tcaagaactt gtatttctct gccaatgctt tcaatggaac gacagtaccc tcataactga
     4381 aatcatccaa gatatcaacg tgtgttttca aaaggcaaat gtatggaccc aacgtttcaa
     4441 caagtttcaa tagctcatca gtcgaacgaa cgtcaagaga agcacacaaa ttggtcttct
     4501 tttcatccat taaacgtaaa agtttcgatg caaccggact tgcatgagtc tcagctctac
     4561 tggtatatga ttttgtggac atggtgcaac taattgacgg gagtgtattg acgctggcgt
     4621 actggctttc acaaaatggc ccaatcacaa ccacatctta gatagttgaa atgactttag
     4681 ataacatcaa ttgagatgag cttaatcatg tcaaagctaa aagtgtcacc atgaacgaca
     4741 attcttaagc aaatcacgtg atatagatcc acgaataacc accatttgat gctcgaggca
     4801 agtaatgtgt gtaaaaaaat gcgttaccac catccaatgc agaccgatct tctacccaga
     4861 atcacatata tttatgtacc gagtaccttt tttctatctt ccaattgctt ctcccatatg
     4921 attgtctccg taagctcgaa atttctaagt tggattttaa tcttcacgca ggatgacagt
     4981 tcgatgagct tctgaggagt gtttagaaca taatcattta tccatggtct atctcttctt
     5041 gtcgcttttt ctcctcgata gaacctaaat aaaacgagct ctcgagaacc cttaatataa
     5101 cttcgtataa tgtatgctat acgaagttat taggtgatat cagatccact agtggcctat
     5161 gcggccgcgg atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt
     5221 aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5281 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5341 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5401 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5461 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5521 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5581 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5641 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5701 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5761 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5821 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5881 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5941 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6001 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6061 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6121 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6181 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6241 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6301 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6361 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6421 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6481 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6541 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6601 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6661 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6721 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6781 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6841 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6901 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6961 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     7021 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7081 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7141 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7201 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     7261 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     7321 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     7381 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     7441 cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa
     7501 tacataacct tatgtatcat acacatacga tttaggtgac actata
//
