LOCUS       sequence-461670-        4895 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 4895)
  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 4895)
  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-HIS3
FEATURES             Location/Qualifiers
     source          1..4895
                     /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        1183..1526
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1527..2225
                     /codon_start=1
                     /product="S. kluyveri imidazoleglycerol phosphate 
                     dehydratase"
                     /label=k-HIS3
                     /translation="MAEPAQKKQKQTVQERKAFISRITNETKIQIAISLNGGYIQIKDS
                     ILPAKKDDDVASQATQSQVIDIHTGVGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHT
                     TEDCGIALGQAFKEAMGAVRGVKRFGTGFAPLDEALSRAVVDLSSRPFAVIDLGLKREM
                     IGDLSTEMIPHFLESFAEAARITLHVDCLRGFNDHHRSESAFKALAVAIREAISSNGTN
                     DVPSTKGVLM"
     terminator      2229..2426
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2530..2549)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2531..2549)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2636..2653)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2807..3395)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2887..2906)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3566..4357,4358..4426))
                     /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     4189..4208
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4427..4531)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4599..4617
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4655..4677)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4777..4796
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4877..4895
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4877..4894
                     /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 tctgtttagc ttgcctcgtc cccgccgggt cacccggcca gcgacatgga ggcccagaat
     1201 accctccttg acagtcttga cgtgcgcagc tcaggggcat gatgtgactg tcgcccgtac
     1261 atttagccca tacatcccca tgtataatca tttgcatcca tacattttga tggccgcacg
     1321 gcgcgaagca aaaattacgg ctcctcgctg cagacctgcg agcagggaaa cgctcccctc
     1381 acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga gaaatataaa aggttaggat
     1441 ttgccactga ggttcttctt tcatatactt ccttttaaaa tcttgctagg atacagttct
     1501 cacatcacat ccgaacataa acaaccatgg cagaaccagc ccaaaaaaag caaaaacaaa
     1561 ctgttcagga gcgcaaggcg tttatctccc gtatcactaa tgaaactaaa attcaaatcg
     1621 ctatttcgct gaatggtggt tatattcaaa taaaagattc gattcttcct gcaaagaagg
     1681 atgacgatgt agcttcccaa gctactcagt cacaggtcat cgatattcac acaggtgttg
     1741 gctttttgga tcatatgatc catgcgttgg caaaacactc tggttggtct cttattgttg
     1801 aatgtattgg tgacctgcac attgacgatc accatactac cgaagattgc ggtatcgcat
     1861 tagggcaagc gttcaaagaa gcaatgggtg ctgtccgtgg tgtaaaaaga ttcggtactg
     1921 ggttcgcacc attggatgag gcgctatcac gtgccgtagt cgatttatct agtagaccat
     1981 ttgctgtaat cgaccttgga ttgaagagag agatgattgg tgatttatcc actgaaatga
     2041 ttccacactt tttggaaagt ttcgcggagg cggccagaat tactttgcat gttgattgtc
     2101 tgagaggttt caacgatcac cacagaagtg agagtgcgtt caaggctttg gctgttgcca
     2161 taagagaagc tatttctagc aatggcacca atgacgttcc ctcaaccaaa ggtgttttga
     2221 tgtgaagtac tgacaataaa aagattcttg ttttcaagaa cttgtcattt gtatagtttt
     2281 tttatattgt agttgttcta ttttaatcaa atgttagcgt gatttatatt ttttttcgcc
     2341 tcgacatcat ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca tgcgtcaatc
     2401 gtatgtgaat gctggtcgct atactgctgt cgattcgata ctaacgccgc catccagttt
     2461 aaacgagctc gaattcatcg atgatatcag atccactagt ggcctatgcg gccgcggatc
     2521 tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac ctgcattaat
     2581 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     2641 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     2701 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     2761 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     2821 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     2881 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2941 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3001 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3061 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3121 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3181 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3241 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3301 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3361 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3421 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3481 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3541 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3601 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3661 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3721 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3781 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3841 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3901 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3961 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4021 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4081 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4141 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4201 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4261 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4321 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4381 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4441 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4501 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     4561 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     4621 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     4681 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     4741 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
     4801 gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac ataaccttat
     4861 gtatcataca catacgattt aggtgacact ataga
//
