LOCUS       sequence-424583-        6258 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone plasmid containing ADH3 promoter and 4V5-APEX2 tag. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6258)
  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 6258)
  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-pADH3-4V5-APEX2-URA
FEATURES             Location/Qualifiers
     source          1..6258
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1072..1113
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1120..1161
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1168..1209
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1216..1257
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1264..2010
                     /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             2017..2043
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      2085..2272
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2140..2164)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    2324..2357
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3809..3842
                     /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(3895..3914)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3896..3914)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4001..4018)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4172..4760)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4252..4271)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4931..5722,5723..5791))
                     /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     5554..5573
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5792..5896)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5964..5982
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6020..6042)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6142..6161
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6242..6258,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6242..6258,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gctttcctta cgaaaaaaaa gatcaaggaa acatgtcgga
       61 aacaaatggg aaacaacaaa aaggacttat cattcctact tttttgtcga gggcatttgt
      121 cgagggtaat actgcatact gcctattgtc ggtcccgcag aattaatagt ggctgctcat
      181 acacaacgaa aaaaattgtt aacaaaatag aaggattcgt ctatgccctt ttttcagtgc
      241 ggaaattata taccacacaa gccgtcgaag cccatagggt cctttaacaa taaagactct
      301 cctaaaaaaa tttttcttgt gacacgtcag tgcaggatat aaatgtagca agcgtatata
      361 acaaatgaca aagggttctt gcgcaaggtt actttagcgt tgctggataa tgatcggatt
      421 gtttttagat cacgatagca catttgagga atcgtatcga aacggacagg gcaagagtac
      481 gagggcgaaa tgaacttata acaccattga tattttctcc tcaatcttat cattccacgc
      541 acattatttt cctactatgt tatgcgggaa cagttcctaa ggaatttttc taatggcatt
      601 tcggcagcgt catcaaatgt acgcacccac ttcaagtgat cattcgctcg ttactaccta
      661 tcacgtgtcg catcactcgc gacgtccatg cgcatgctgt gccgcaggag aattcgagat
      721 gcgaatgagc agcagccatt ttgatgttgt gagcatcgga acgtttctgc gtccgtacac
      781 tgtccttttg ttacttagat aatggctaag gcaagcagtc cgggccacag gagtcaaagg
      841 cttttcgcca gctcctaaac gctggaagtg taattttttt tcgtcttata aaattaaaca
      901 aaccctttta gaaaggaaca ctcgctttat ctcttcgacc gaatttacta tacatggata
      961 tatatatatt atcttctgtt cacagttaaa actaggaata gtatagtcat aagttaacac
     1021 catccgtacg ctgcaggtcg tgggattcct gggttaatta acatcgctag cggtaaacca
     1081 attccaaatc cattgttggg tttggattct actcctagcg gtaaaccaat tccaaatcca
     1141 ttgttgggtt tggattctac tcctagcggt aaaccaattc caaatccatt gttgggtttg
     1201 gattctactc ctagcggtaa accaattcca aatccattgt tgggtttgga ttctactcct
     1261 aggggaaagt cttacccaac tgtgagtgct gattaccagg acgccgttga gaaggcgaag
     1321 aagaagctca gaggcttcat cgctgagaag agatgcgctc ctctaatgct ccgtttggca
     1381 ttccactctg ctggaacctt tgacaagggc acgaagaccg gtggaccctt cggaaccatc
     1441 aagcaccctg ccgaactggc tcacagcgct aacaacggtc ttgacatcgc tgttaggctt
     1501 ttggagccac tcaaggcgga gttccctatt ttgagctacg ccgatttcta ccagttggct
     1561 ggcgttgttg ccgttgaggt cacgggtgga cctaaggttc cattccaccc tggaagagag
     1621 gacaagcctg agccaccacc agagggtcgc ttgcccgatc ccactaaggg ttctgaccat
     1681 ttgagagatg tgtttggcaa agctatgggg cttactgacc aagatatcgt tgctctatct
     1741 gggggtcaca ctattggagc tgcacacaag gagcgttctg gatttgaggg tccctggacc
     1801 tctaatcctc ttattttcga caactcatac ttcacggagt tgttgagtgg tgagaaggaa
     1861 ggtctccttc agctaccttc tgacaaggct cttttgtctg accctgtatt ccgccctctc
     1921 gttgacaaat atgcagcgga cgaagatgcc ttctttgctg attacgctga ggctcaccaa
     1981 aagctttccg agcttgggtt tgctgatgcc ctgcagctgc ctcccctgga gcgcctgacc
     2041 ctggacgctc agtgcagatc ttgaggcgcg ccacttctaa ataagcgaat ttcttatgat
     2101 ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa ttttaaagtg
     2161 actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg taggtcaggt
     2221 tgctttctca ggtatagtat gaggtcgctc ttattgacca cacctctacc ggcagatccg
     2281 ctagggataa cagggtaata tctgcaggtc gacaaccctt aatataactt cgtataatgt
     2341 atgctatacg aagttattag gtctagagat cccaatacaa cagatcacgt gatctttttg
     2401 taagatgaag ttgaagtagt gttgcaccgt gccaatgcag gtggctatta gattaaatat
     2461 gtgatttgtt ctattaagtt tcctgtataa ttaatgggga gcgctgattc tcttttggta
     2521 cgcttcccat ccagcatttc tgtatctttc accttcaacc ttaggatctc tacccttggc
     2581 gaaaagtcct ctgccaacaa tgatgatatc tgatccacca cttacaactt cgtcgacggt
     2641 tctgtactgc tgacccaatg catcgccttt gtcgtctaaa cctacacctg gggtcatgat
     2701 tagccaatca aacccttctt ctcttcctcc catatcgttc tgagcaatga acccaataac
     2761 gaaatcttta tcactctttg caatatcaac ggtaccctta gtatattcac cgtgtgctag
     2821 agaacccttg gaagacaatt cagcaagcat caataatccc cttggttctt tggtgacctc
     2881 ttgcgcacct tgtttcaagc cagcaacaat accagcacca gtaaccccgt gggcgttggt
     2941 gatatcagac cattctgcga tacggtaaac gcccgatgta tattgtaatt tgactgtgtt
     3001 accgatatcg gcgaattttc tgtcctcaaa tatcaagaac ttgtatttct ctgccaatgc
     3061 tttcaatgga acgacagtac cctcataact gaaatcatcc aagatatcaa cgtgtgtttt
     3121 caaaaggcaa atgtatggac ccaacgtttc aacaagtttc aatagctcat cagtcgaacg
     3181 aacgtcaaga gaagcacaca aattggtctt cttttcatcc attaaacgta aaagtttcga
     3241 tgcaaccgga cttgcatgag tctcagctct actggtatat gattttgtgg acatggtgca
     3301 actaattgac gggagtgtat tgacgctggc gtactggctt tcacaaaatg gcccaatcac
     3361 aaccacatct tagatagttg aaatgacttt agataacatc aattgagatg agcttaatca
     3421 tgtcaaagct aaaagtgtca ccatgaacga caattcttaa gcaaatcacg tgatatagat
     3481 ccacgaataa ccaccatttg atgctcgagg caagtaatgt gtgtaaaaaa atgcgttacc
     3541 accatccaat gcagaccgat cttctaccca gaatcacata tatttatgta ccgagtacct
     3601 tttttctatc ttccaattgc ttctcccata tgattgtctc cgtaagctcg aaatttctaa
     3661 gttggatttt aatcttcacg caggatgaca gttcgatgag cttctgagga gtgtttagaa
     3721 cataatcatt tatccatggt ctatctcttc ttgtcgcttt ttctcctcga tagaacctaa
     3781 ataaaacgag ctctcgagaa cccttaatat aacttcgtat aatgtatgct atacgaagtt
     3841 attaggtgat atcagatcca ctagtggcct atgcggccgc ggatctgccg gtctccctat
     3901 agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc ggccaacgcg
     3961 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4021 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4081 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4141 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4201 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4261 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4321 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4381 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4441 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4501 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4561 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4621 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4681 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4741 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4801 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4861 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4921 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4981 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5041 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5101 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5161 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5221 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5281 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5341 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5401 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5461 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5521 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5581 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5641 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5701 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5761 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5821 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5881 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     5941 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     6001 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     6061 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     6121 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgg
     6181 acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac
     6241 gatttaggtg acactata
//
