LOCUS       sequence-422540-        6720 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing p500-Erg6-V5-APEX2. Stains cell peripheral in addition to
            lipid droplets..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6720)
  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 6720)
  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-p500-Erg6-V5-APEX2-URA
FEATURES             Location/Qualifiers
     source          1..6720
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1675..1716
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1717..2463
                     /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             2470..2496
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      2547..2734
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2602..2626)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    2786..2819
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4271..4304
                     /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(4357..4376)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4358..4376)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4463..4480)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4634..5222)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4714..4733)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5393..6184,6185..6253))
                     /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     6016..6035
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6254..6358)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6426..6444
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6482..6504)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6604..6623
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6704..6720,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6704..6720,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcttact atttttgaaa agatgcaaca gggtaagatc
       61 agcaaaattt agggccgaac aaagccccga tcttcgtata tggtacctcg ttcccgtact
      121 tattttcaat ttttggcgtc gtttactgcc gtttccgata aatacaaaaa agttttcgag
      181 aagcgagagt aagtgaggag cagacaatgt attattcact gtaaactgct ccacttcgtc
      241 tcaatggtac acttcatttt tatcgcctta cgaagcatgc gttttatgcg aagattggtg
      301 agaaacctcc aatacttgct gttgccgata acttcttcat tgctttttat atagttcggg
      361 tgttttctcc tatcctctgc tgctctcttt ttctttccat tcgtctttgt aatttataag
      421 ctgttaattt tcattcaagt ttcccttatc tgttttactt tcgatttaag ttttacataa
      481 tttaaaaaaa caagaataaa ataataatat agtaggcagc ataagatgag tgaaacagaa
      541 ttgagaaaaa gacaggccca attcactagg gagttacatg gtgatgatat tggtaaaaag
      601 acaggtttga gtgcattgat gtcgaagaac aactctgccc aaaaggaagc cgttcagaag
      661 tacttgagaa attgggatgg tagaaccgat aaagatgccg aagaacgtcg tcttgaggat
      721 tataatgaag ccacacattc ctactataac gtcgttacag atttctatga atatggttgg
      781 ggttcctctt tccatttcag cagattttat aaaggtgaga gtttcgctgc ctcgatagca
      841 agacatgaac attatttagc ttacaaggct ggtattcaaa gaggcgattt agttctcgac
      901 gttggttgtg gtgttggggg cccagcaaga gagattgcaa gatttaccgg ttgtaacgtc
      961 atcggtctaa acaataacga ttaccaaatt gccaaggcaa aatattacgc taaaaaatac
     1021 aatttgagtg accaaatgga ctttgtaaag ggtgatttca tgaaaatgga tttcgaagaa
     1081 aacactttcg acaaagttta tgcaattgag gccacatgtc acgctccaaa attagaaggt
     1141 gtatacagcg aaatctacaa ggttttgaaa ccgggtggta cctttgctgt ttacgaatgg
     1201 gtaatgactg ataaatatga cgaaaacaat cctgaacata gaaagatcgc ttatgaaatt
     1261 gaactaggtg atggtatccc aaagatgttc catgtcgacg tggctaggaa agcattgaag
     1321 aactgtggtt tcgaagtcct cgttagcgaa gacctggcgg acaatgatga tgaaatccct
     1381 tggtattacc cattaactgg tgagtggaag tacgttcaaa acttagctaa tttggccaca
     1441 tttttcagaa cttcttactt gggtagacaa tttactacag caatggttac tgtaatggaa
     1501 aaattaggtc tagccccaga aggttccaag gaagttactg ctgctctaga aaatgctgcg
     1561 gttggtttag ttgccggtgg taagtccaag ttattcactc caatgatgct tttcgtcgct
     1621 aggaagccag aaaacgccga aaccccctcc caaacttccc aagaagcaac tcaaggtaaa
     1681 ccaattccaa atccattgtt gggtttggat tctactggaa agtcttaccc aactgtgagt
     1741 gctgattacc aggacgccgt tgagaaggcg aagaagaagc tcagaggctt catcgctgag
     1801 aagagatgcg ctcctctaat gctccgtttg gcattccact ctgctggaac ctttgacaag
     1861 ggcacgaaga ccggtggacc cttcggaacc atcaagcacc ctgccgaact ggctcacagc
     1921 gctaacaacg gtcttgacat cgctgttagg cttttggagc cactcaaggc ggagttccct
     1981 attttgagct acgccgattt ctaccagttg gctggcgttg ttgccgttga ggtcacgggt
     2041 ggacctaagg ttccattcca ccctggaaga gaggacaagc ctgagccacc accagagggt
     2101 cgcttgcccg atcccactaa gggttctgac catttgagag atgtgtttgg caaagctatg
     2161 gggcttactg accaagatat cgttgctcta tctgggggtc acactattgg agctgcacac
     2221 aaggagcgtt ctggatttga gggtccctgg acctctaatc ctcttatttt cgacaactca
     2281 tacttcacgg agttgttgag tggtgagaag gaaggtctcc ttcagctacc ttctgacaag
     2341 gctcttttgt ctgaccctgt attccgccct ctcgttgaca aatatgcagc ggacgaagat
     2401 gccttctttg ctgattacgc tgaggctcac caaaagcttt ccgagcttgg gtttgctgat
     2461 gccctgcagc tgcctcccct ggagcgcctg accctggacg ctcagtgcag atcttagcct
     2521 aggtgaggcg cgccacttct aaataagcga atttcttatg atttatgatt tttattatta
     2581 aataagttat aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac
     2641 gaaaattctt attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagt
     2701 atgaggtcgc tcttattgac cacacctcta ccggcagatc cgctagggat aacagggtaa
     2761 tatctgcagg tcgacaaccc ttaatataac ttcgtataat gtatgctata cgaagttatt
     2821 aggtctagag atcccaatac aacagatcac gtgatctttt tgtaagatga agttgaagta
     2881 gtgttgcacc gtgccaatgc aggtggctat tagattaaat atgtgatttg ttctattaag
     2941 tttcctgtat aattaatggg gagcgctgat tctcttttgg tacgcttccc atccagcatt
     3001 tctgtatctt tcaccttcaa ccttaggatc tctacccttg gcgaaaagtc ctctgccaac
     3061 aatgatgata tctgatccac cacttacaac ttcgtcgacg gttctgtact gctgacccaa
     3121 tgcatcgcct ttgtcgtcta aacctacacc tggggtcatg attagccaat caaacccttc
     3181 ttctcttcct cccatatcgt tctgagcaat gaacccaata acgaaatctt tatcactctt
     3241 tgcaatatca acggtaccct tagtatattc accgtgtgct agagaaccct tggaagacaa
     3301 ttcagcaagc atcaataatc cccttggttc tttggtgacc tcttgcgcac cttgtttcaa
     3361 gccagcaaca ataccagcac cagtaacccc gtgggcgttg gtgatatcag accattctgc
     3421 gatacggtaa acgcccgatg tatattgtaa tttgactgtg ttaccgatat cggcgaattt
     3481 tctgtcctca aatatcaaga acttgtattt ctctgccaat gctttcaatg gaacgacagt
     3541 accctcataa ctgaaatcat ccaagatatc aacgtgtgtt ttcaaaaggc aaatgtatgg
     3601 acccaacgtt tcaacaagtt tcaatagctc atcagtcgaa cgaacgtcaa gagaagcaca
     3661 caaattggtc ttcttttcat ccattaaacg taaaagtttc gatgcaaccg gacttgcatg
     3721 agtctcagct ctactggtat atgattttgt ggacatggtg caactaattg acgggagtgt
     3781 attgacgctg gcgtactggc tttcacaaaa tggcccaatc acaaccacat cttagatagt
     3841 tgaaatgact ttagataaca tcaattgaga tgagcttaat catgtcaaag ctaaaagtgt
     3901 caccatgaac gacaattctt aagcaaatca cgtgatatag atccacgaat aaccaccatt
     3961 tgatgctcga ggcaagtaat gtgtgtaaaa aaatgcgtta ccaccatcca atgcagaccg
     4021 atcttctacc cagaatcaca tatatttatg taccgagtac cttttttcta tcttccaatt
     4081 gcttctccca tatgattgtc tccgtaagct cgaaatttct aagttggatt ttaatcttca
     4141 cgcaggatga cagttcgatg agcttctgag gagtgtttag aacataatca tttatccatg
     4201 gtctatctct tcttgtcgct ttttctcctc gatagaacct aaataaaacg agctctcgag
     4261 aacccttaat ataacttcgt ataatgtatg ctatacgaag ttattaggtg atatcagatc
     4321 cactagtggc ctatgcggcc gcggatctgc cggtctccct atagtgagtc gtattaattt
     4381 cgataagcca ggttaacctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4441 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4501 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4561 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4621 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4681 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4741 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4801 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4861 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4921 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4981 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5041 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5101 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5161 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5221 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5281 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5341 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5401 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5461 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5521 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5581 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5641 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5701 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5761 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5821 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5881 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5941 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6001 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6061 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6121 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6181 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6241 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6301 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6361 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     6421 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     6481 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     6541 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     6601 atgcggcatc agagcagatt gtactgagag tgcaccatat ggacatattg tcgttagaac
     6661 gcggctacaa ttaatacata accttatgta tcatacacat acgatttagg tgacactata
//
