LOCUS       sequence-461672-        5836 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template plasmid for the endogenous tagging with FLAG-APEX2-NAS in 
            yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5836)
  AUTHORS   Filali-Mouncef Y, Leytens A, Vargas Duarte P, Zampieri M, Dengjel J,
            Reggiori F
  TITLE     An APEX2-based proximity-dependent biotinylation assay with temporal
            specificity to study protein interactions during autophagy in the 
            yeast Saccharomyces cerevisiae.
  JOURNAL   Autophagy. 2024 Oct;20(10):2323-2337. doi: 
            10.1080/15548627.2024.2366749. Epub 2024 Jul 3.
  PUBMED    38958087
REFERENCE   2  (bases 1 to 5836)
  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"; doi: 
            "10.1080/15548627.2024.2366749"; journalName: "Autophagy"; date: 
            "2024-10"; volume: "20"; issue: "10"; pages: "2323-2337"
COMMENT     Sequence Label: pFA6a-FLAG-APEX2-NES-K.l.LEU2
FEATURES             Location/Qualifiers
     source          1..5836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..87
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             88..834
                     /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             841..867
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      921..1108
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(976..1000)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3471..3490)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3472..3490)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3577..3594)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3748..4336)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3828..3847)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4507..5298,5299..5367))
                     /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     5130..5149
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5368..5472)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5540..5558
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5596..5618)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5718..5737
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5818..5836
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5818..5835
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 acgcggccgc cagctgaagc ttcgtacgct gcaggtcgac ggatccccgg gttaattaat
       61 atggactaca aggatgacga cgataaggga aagtcttacc caactgtgag tgctgattac
      121 caggacgccg ttgagaaggc gaagaagaag ctcagaggct tcatcgctga gaagagatgc
      181 gctcctctaa tgctccgttt ggcattccac tctgctggaa cctttgacaa gggcacgaag
      241 accggtggac ccttcggaac catcaagcac cctgccgaac tggctcacag cgctaacaac
      301 ggtcttgaca tcgctgttag gcttttggag ccactcaagg cggagttccc tattttgagc
      361 tacgccgatt tctaccagtt ggctggcgtt gttgccgttg aggtcacggg tggacctaag
      421 gttccattcc accctggaag agaggacaag cctgagccac caccagaggg tcgcttgccc
      481 gatcccacta agggttctga ccatttgaga gatgtgtttg gcaaagctat ggggcttact
      541 gaccaagata tcgttgctct atctgggggt cacactattg gagctgcaca caaggagcgt
      601 tctggatttg agggtccctg gacctctaat cctcttattt tcgacaactc atacttcacg
      661 gagttgttga gtggtgagaa ggaaggtctc cttcagctac cttctgacaa ggctcttttg
      721 tctgaccctg tattccgccc tctcgttgac aaatatgcag cggacgaaga tgccttcttt
      781 gctgattacg ctgaggctca ccaaaagctt tccgagcttg ggtttgctga tgccctgcag
      841 ctgcctcccc tggagcgcct gaccctggac ggatccggtg gaggccctgc aggggcttag
      901 ggcgcgccac ttctaaataa gcgaatttct tatgatttat gatttttatt attaaataag
      961 ttataaaaaa aataagtgta tacaaatttt aaagtgactc ttaggtttta aaacgaaaat
     1021 tcttattctt gagtaactct ttcctgtagg tcaggttgct ttctcaggta tagtatgagg
     1081 tcgctcttat tgaccacacc tctaccggca gatccgctag ggataacagg gtaatataga
     1141 tccgctgtga agatcccagc aaaggcttac aaagtgttat ctcttttgag acttgttgag
     1201 ttgaacactg gtgttttcat caaacttacc aaggacgtgt acccattgtt gaaacttgta
     1261 tcaccatata ttgttatcgg acaaccttca cttgcatcta tccgttcttt aatccaaaag
     1321 agatctagaa taatgtggca aaggccagaa gataaagaac caaaagagat aatcttgaat
     1381 gacaacaata tcgttgaaga gaaattaggt gatgaaggtg tcatttgtat cgaggatatc
     1441 atccatgaga tttcgacgtt gggcgaaaat ttctcgaaat gtactttctt cctattacca
     1501 ttcaaattga acagagaagt cagtggattc ggtgccatct cccgtttgaa taaactgaaa
     1561 atgcgcgaac aaaacaagaa gactcgtcaa atttcaaacg ctgccacggc tccagttatc
     1621 caagtagata tcgactcaat gatttccaag ttgaattgat taactataaa aggaaaatat
     1681 ctgtacaata gacatcgggc tcccattggc cctacccaca tatgtagaaa tacattactc
     1741 tattcactac tgcatttagt tatgtttaac atttgatata gcagacgcta ccgccaggca
     1801 caatatattc cccttccctc ttgccattcg ctgtacttgt ggtagattcc aattcagcgc
     1861 agtcacgtgc tagtaatcac cgcatttttt tcttttcctt tcaggctaaa accggttccg
     1921 ggcctgatcc ctgcactcat tttctaacgg aaaaccttca gaagcataac tacccattcc
     1981 agtttagagt catgacaggt tcaacatcag atgcttcata tacttttata tattgaatta
     2041 tataaatata tctatgtact ctaagtaagt acatctgctt taacgcattc ctaaatttgc
     2101 ttcgatttat ttttattgtt gatacctatt tgaagaagta aaaagtatcc cacactacac
     2161 agattatacc atgtctaaga atatcgttgt cctaccgggt gatcacgtcg gtaaagaagt
     2221 tactgacgaa gctattaagg tcttgaatgc cattgctgaa gtccgtccag aaattaagtt
     2281 caatttccaa catcacttga tcgggggtgc tgccatcgat gccactggca ctcctttacc
     2341 agatgaagct ctagaagcct ctaagaaagc cgatgctgtc ttactaggtg ctgttggtgg
     2401 tccaaaatgg ggtacgggcg cagttagacc agaacaaggt ctattgaaga tcagaaagga
     2461 attgggtcta tacgccaact tgagaccatg taactttgct tctgattctt tactagatct
     2521 ttctcctttg aagcctgaat atgcaaaggg taccgatttc gtcgtcgtta gagaattggt
     2581 tggtggtatc tactttggtg aaagaaaaga agatgaaggt gacggagttg cttgggactc
     2641 tgagaaatac agtgttcctg aagttcaaag aattacaaga atggctgctt tcttggcatt
     2701 gcaacaaaac ccaccattac caatctggtc tcttgacaag gctaacgtgc ttgcctcttc
     2761 cagattgtgg agaaagactg ttgaagaaac catcaagact gagttcccac aattaactgt
     2821 tcagcaccaa ttgatcgact ctgctgctat gattttggtt aaatcaccaa ctaagctaaa
     2881 cggtgttgtt attaccaaca acatgtttgg tgatattatc tccgatgaag cctctgttat
     2941 tccaggttct ttgggtttat taccttctgc atctctagct tccctacctg acactaacaa
     3001 ggcattcggt ttgtacgaac catgtcatgg ttctgcccca gatttaccag caaacaaggt
     3061 taacccaatt gctaccatct tatctgcagc tatgatgttg aagttatcct tggatttggt
     3121 tgaagaaggt agggctcttg aagaagctgt tagaaatgtc ttggatgcag gtgtcagaac
     3181 cggtgacctt ggtggttcta actctaccac tgaggttggc gatgctatcg ccaaggctgt
     3241 caaggaaatc ttggcttaaa gagtcttttg taacgacccc gtctccacca acttggtatg
     3301 cttgaaatct caaggccatt acacattcag ttatgtgaac gaaaggtctt tatttaacgt
     3361 agcataaact aaataataca ggttccggtt agcctgcgtt taaacgagct cgaattcatc
     3421 gatgatatca gatccactag tggcctatgc ggccgcggat ctgccggtct ccctatagtg
     3481 agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg
     3541 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3601 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3661 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3721 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3781 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3841 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3901 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3961 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4021 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4081 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4141 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4201 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4261 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4321 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4381 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4441 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4501 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4561 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4621 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4681 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4741 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4801 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4861 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4921 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4981 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5041 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5101 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5161 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5221 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5281 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5341 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5401 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5461 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     5521 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     5581 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     5641 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     5701 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatggacat
     5761 attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt
     5821 taggtgacac tataga
//
