LOCUS       sequence-424591-        5258 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For ORF tagging with 4V5-APEX2 .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5258)
  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 5258)
  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-4V5-APEX2-URA
FEATURES             Location/Qualifiers
     source          1..5258
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             72..113
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             120..161
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             168..209
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             216..257
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             264..1010
                     /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             1017..1043
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      1085..1272
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1140..1164)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    1324..1357
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2809..2842
                     /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(2895..2914)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2896..2914)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3001..3018)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3172..3760)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3252..3271)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3931..4722,4723..4791))
                     /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     4554..4573
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4792..4896)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4964..4982
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5020..5042)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5142..5161
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5242..5258,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5242..5258,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg tgggattcct gggttaatta
       61 acatcgctag cggtaaacca attccaaatc cattgttggg tttggattct actcctagcg
      121 gtaaaccaat tccaaatcca ttgttgggtt tggattctac tcctagcggt aaaccaattc
      181 caaatccatt gttgggtttg gattctactc ctagcggtaa accaattcca aatccattgt
      241 tgggtttgga ttctactcct aggggaaagt cttacccaac tgtgagtgct gattaccagg
      301 acgccgttga gaaggcgaag aagaagctca gaggcttcat cgctgagaag agatgcgctc
      361 ctctaatgct ccgtttggca ttccactctg ctggaacctt tgacaagggc acgaagaccg
      421 gtggaccctt cggaaccatc aagcaccctg ccgaactggc tcacagcgct aacaacggtc
      481 ttgacatcgc tgttaggctt ttggagccac tcaaggcgga gttccctatt ttgagctacg
      541 ccgatttcta ccagttggct ggcgttgttg ccgttgaggt cacgggtgga cctaaggttc
      601 cattccaccc tggaagagag gacaagcctg agccaccacc agagggtcgc ttgcccgatc
      661 ccactaaggg ttctgaccat ttgagagatg tgtttggcaa agctatgggg cttactgacc
      721 aagatatcgt tgctctatct gggggtcaca ctattggagc tgcacacaag gagcgttctg
      781 gatttgaggg tccctggacc tctaatcctc ttattttcga caactcatac ttcacggagt
      841 tgttgagtgg tgagaaggaa ggtctccttc agctaccttc tgacaaggct cttttgtctg
      901 accctgtatt ccgccctctc gttgacaaat atgcagcgga cgaagatgcc ttctttgctg
      961 attacgctga ggctcaccaa aagctttccg agcttgggtt tgctgatgcc ctgcagctgc
     1021 ctcccctgga gcgcctgacc ctggacgctc agtgcagatc ttgaggcgcg ccacttctaa
     1081 ataagcgaat ttcttatgat ttatgatttt tattattaaa taagttataa aaaaaataag
     1141 tgtatacaaa ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa
     1201 ctctttcctg taggtcaggt tgctttctca ggtatagtat gaggtcgctc ttattgacca
     1261 cacctctacc ggcagatccg ctagggataa cagggtaata tctgcaggtc gacaaccctt
     1321 aatataactt cgtataatgt atgctatacg aagttattag gtctagagat cccaatacaa
     1381 cagatcacgt gatctttttg taagatgaag ttgaagtagt gttgcaccgt gccaatgcag
     1441 gtggctatta gattaaatat gtgatttgtt ctattaagtt tcctgtataa ttaatgggga
     1501 gcgctgattc tcttttggta cgcttcccat ccagcatttc tgtatctttc accttcaacc
     1561 ttaggatctc tacccttggc gaaaagtcct ctgccaacaa tgatgatatc tgatccacca
     1621 cttacaactt cgtcgacggt tctgtactgc tgacccaatg catcgccttt gtcgtctaaa
     1681 cctacacctg gggtcatgat tagccaatca aacccttctt ctcttcctcc catatcgttc
     1741 tgagcaatga acccaataac gaaatcttta tcactctttg caatatcaac ggtaccctta
     1801 gtatattcac cgtgtgctag agaacccttg gaagacaatt cagcaagcat caataatccc
     1861 cttggttctt tggtgacctc ttgcgcacct tgtttcaagc cagcaacaat accagcacca
     1921 gtaaccccgt gggcgttggt gatatcagac cattctgcga tacggtaaac gcccgatgta
     1981 tattgtaatt tgactgtgtt accgatatcg gcgaattttc tgtcctcaaa tatcaagaac
     2041 ttgtatttct ctgccaatgc tttcaatgga acgacagtac cctcataact gaaatcatcc
     2101 aagatatcaa cgtgtgtttt caaaaggcaa atgtatggac ccaacgtttc aacaagtttc
     2161 aatagctcat cagtcgaacg aacgtcaaga gaagcacaca aattggtctt cttttcatcc
     2221 attaaacgta aaagtttcga tgcaaccgga cttgcatgag tctcagctct actggtatat
     2281 gattttgtgg acatggtgca actaattgac gggagtgtat tgacgctggc gtactggctt
     2341 tcacaaaatg gcccaatcac aaccacatct tagatagttg aaatgacttt agataacatc
     2401 aattgagatg agcttaatca tgtcaaagct aaaagtgtca ccatgaacga caattcttaa
     2461 gcaaatcacg tgatatagat ccacgaataa ccaccatttg atgctcgagg caagtaatgt
     2521 gtgtaaaaaa atgcgttacc accatccaat gcagaccgat cttctaccca gaatcacata
     2581 tatttatgta ccgagtacct tttttctatc ttccaattgc ttctcccata tgattgtctc
     2641 cgtaagctcg aaatttctaa gttggatttt aatcttcacg caggatgaca gttcgatgag
     2701 cttctgagga gtgtttagaa cataatcatt tatccatggt ctatctcttc ttgtcgcttt
     2761 ttctcctcga tagaacctaa ataaaacgag ctctcgagaa cccttaatat aacttcgtat
     2821 aatgtatgct atacgaagtt attaggtgat atcagatcca ctagtggcct atgcggccgc
     2881 ggatctgccg gtctccctat agtgagtcgt attaatttcg ataagccagg ttaacctgca
     2941 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3001 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3061 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3121 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3181 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3241 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3301 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3361 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3421 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3481 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3541 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3601 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3661 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3721 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3781 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3841 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3901 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3961 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4021 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4081 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4141 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4201 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4261 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4321 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4381 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4441 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4501 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4561 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4621 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4681 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4741 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4801 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     4861 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     4921 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     4981 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     5041 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     5101 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     5161 actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt aatacataac
     5221 cttatgtatc atacacatac gatttaggtg acactata
//
