LOCUS       sequence-461668-        4771 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 4771)
  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 4771)
  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-NatMX6
FEATURES             Location/Qualifiers
     source          1..4771
                     /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"
     gene            1183..2302
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        1183..1526
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1527..2099
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      2105..2302
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2406..2425)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2407..2425)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2512..2529)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2683..3271)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2763..2782)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3442..4233,4234..4302))
                     /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     4065..4084
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4303..4407)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4475..4493
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4531..4553)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4653..4672
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4753..4771
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4753..4770
                     /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 cctttttaaa tcttgctagg atacagttct
     1501 cacatcacat ccgaacataa acaaccatgg gtaccactct tgacgacacg gcttaccggt
     1561 accgcaccag tgtcccgggg gacgccgagg ccatcgaggc actggatggg tccttcacca
     1621 ccgacaccgt cttccgcgtc accgccaccg gggacggctt caccctgcgg gaggtgccgg
     1681 tggacccgcc cctgaccaag gtgttccccg acgacgaatc ggacgacgaa tcggacgacg
     1741 gggaggacgg cgacccggac tctcggacgt tcgtcgcgta cggggacgac ggcgacctgg
     1801 cgggcttcgt ggtcgtctcg tactccggct ggaaccgccg gctgaccgtc gaggacatcg
     1861 aggtcgcccc ggagcaccgg gggcacgggg tcgggcgcgc gttgatgggg ctcgcgacgg
     1921 agttcgcccg cgagcggggt gccgggcacc tctggctgga ggtcaccaac gtcaacgcac
     1981 cggcgatcca cgcgtaccgg cggatggggt tcaccctctg cggcctggac accgccctgt
     2041 acgacggcac cgcctcggac ggcgagcagg cgctctacat gagcatgccc tgcccctaat
     2101 cagtactgac aataaaaaga ttcttgtttt caagaacttg tcatttgtat agttttttta
     2161 tattgtagtt gttctatttt aatcaaatgt tagcgtgatt tatatttttt ttcgcctcga
     2221 catcatctgc ccagatgcga agttaagtgc gcagaaagta atatcatgcg tcaatcgtat
     2281 gtgaatgctg gtcgctatac tgctgtcgat tcgatactaa cgccgccatc cagtttaaac
     2341 gagctcgaat tcatcgatga tatcagatcc actagtggcc tatgcggccg cggatctgcc
     2401 ggtctcccta tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat
     2461 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     2521 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     2581 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     2641 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2701 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2761 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2821 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     2881 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     2941 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3001 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3061 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3121 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3181 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3241 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3301 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3361 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3421 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     3481 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     3541 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     3601 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     3661 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     3721 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     3781 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     3841 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     3901 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     3961 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4021 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4081 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4141 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4201 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     4261 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     4321 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4381 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     4441 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     4501 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     4561 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     4621 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     4681 gcaccatatg gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat
     4741 catacacata cgatttaggt gacactatag a
//
