LOCUS       sequence-461667-        4491 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 4491)
  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 4491)
  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-TRP1
FEATURES             Location/Qualifiers
     source          1..4491
                     /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"
     promoter        1144..1291
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             1292..1966
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     RBS             2046..2054
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(2126..2145)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2127..2145)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2232..2249)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2403..2991)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2483..2502)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3162..3953,3954..4022))
                     /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     3785..3804
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4023..4127)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4195..4213
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4251..4273)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4373..4392
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4473..4491
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4473..4490
                     /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 tctgtacaat cttgatccgg agcttttctt tttttgccga ttaagaatta attcggtcga
     1201 aaaaagaaaa ggagagggcc aagagggagg gcattggtga ctattgagca cgtgagtata
     1261 cgtgattaag cacacaaagg cagcttggag tatgtctgtt attaatttca caggtagttc
     1321 tggtccattg gtgaaagttt gcggcttgca gagcacagag gccgcagaat gtgctctaga
     1381 ttccgatgct gacttgctgg gtattatatg tgtgcccaat agaaagagaa caattgaccc
     1441 ggttattgca aggaaaattt caagtcttgt aaaagcatat aaaaatagtt caggcactcc
     1501 gaaatacttg gttggcgtgt ttcgtaatca acctaaggag gatgttttgg ctctggtcaa
     1561 tgattacggc attgatatcg tccaactgca tggagatgag tcgtggcaag aataccaaga
     1621 gttcctcggt ttgccagtta ttaaaagact cgtatttcca aaagactgca acatactact
     1681 cagtgcagct tcacagaaac ctcattcgtt tattcccttg tttgattcag aagcaggtgg
     1741 gacaggtgaa cttttggatt ggaactcgat ttctgactgg gttggaaggc aagagagccc
     1801 cgaaagctta cattttatgt tagctggtgg actgacgcca gaaaatgttg gtgatgcgct
     1861 tagattaaat ggcgttattg gtgttgatgt aagcggaggt gtggagacaa atggtgtaaa
     1921 agactctaac aaaatagcaa atttcgtcaa aaatgctaag aaataggtta ttactgagta
     1981 gtatttattt aagtattgtt tgtgcacttg ccgatctatg cggtgtgaaa taccgcacag
     2041 atgcgtaagg aggtttaaac gagctcgaat tcatcgatga tatcagatcc actagtggcc
     2101 tatgcggccg cggatctgcc ggtctcccta tagtgagtcg tattaatttc gataagccag
     2161 gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     2221 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     2281 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2341 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2401 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2461 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2521 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2581 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2641 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2701 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2761 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2821 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2881 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2941 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3001 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3061 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3121 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3181 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3241 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3301 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3361 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3421 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3481 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3541 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3601 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3661 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3721 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3781 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3841 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3901 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3961 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4021 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4081 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4141 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     4201 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     4261 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     4321 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     4381 gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat
     4441 taatacataa ccttatgtat catacacata cgatttaggt gacactatag a
//
