LOCUS       sequence-194906-        8353 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FLAG-APEX2 in Saccharomyces cerevisiae under control of 
            GAL1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8353)
  AUTHORS   Hwang J, Espenshade PJ
  TITLE     Proximity-dependent biotin labeling in yeast using the engineered 
            ascorbate peroxidase APEX2.
  JOURNAL   Biochem J. 2016 Jun 7. pii: BCJ20160106.
  PUBMED    27274088
REFERENCE   2  (bases 1 to 8353)
  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: "Biochem J. 
            2016 Jun 7. pii: BCJ20160106."
COMMENT     Sequence Label: pJH124
FEATURES             Location/Qualifiers
     source          1..8353
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        26..130
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(131..199,200..991)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(349..368)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1162..1750
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1651..1670
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1904..1921
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1936..2076
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2002..2021)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2162..2184
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        2376..2816
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    2376..2493
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     2787..2810
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             2840..2863
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2864..3610
                     /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             3617..3643
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      4036..4223
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4091..4115)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    complement(4244..4277)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(4786..5880)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(5893..6297)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      6774..8116
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(7737..7784)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
        1 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
       61 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
      121 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
      181 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
      241 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
      301 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
      361 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
      421 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
      481 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
      541 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
      601 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
      661 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
      721 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
      781 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
      841 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
      901 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
      961 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     1021 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     1081 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     1141 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     1201 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     1261 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     1321 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     1381 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     1441 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     1501 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     1561 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     1621 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     1681 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     1741 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     1801 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     1861 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     1921 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     1981 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     2041 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     2101 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     2161 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagcaa
     2221 cttcttttct ttttttttct ttccatatac atatccatat ctaatcttac ttatatgttg
     2281 tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag
     2341 taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggtg
     2401 acagccctcc gaaggaagac tctcctccgt gcgtcctcgt cttcaccggt cgcgttcctg
     2461 aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct
     2521 tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaataacg
     2581 aatcaaatta acaaccatag gatgataatg cgattagttt tttagcctta tttctggggt
     2641 aattaatcag cgaagcgatg atttttgatc tatcaacaga tatatcaatg ccaaaactgc
     2701 ataaccactt taactaatac tttcaacatt ttcagtttgt attacttctt attcaaatgt
     2761 cataaaagta tcaacaaaaa attgttaata tacctctata ctttaacgtc aaggagaaaa
     2821 aacaagcttt gcaaagatgg actacaagga tgacgacgat aagggaaagt cttacccaac
     2881 tgtgagtgct gattaccagg acgccgttga gaaggcgaag aagaagctca gaggcttcat
     2941 cgctgagaag agatgcgctc ctctaatgct ccgtttggca ttccactctg ctggaacctt
     3001 tgacaagggc acgaagaccg gtggaccctt cggaaccatc aagcaccctg ccgaactggc
     3061 tcacagcgct aacaacggtc ttgacatcgc tgttaggctt ttggagccac tcaaggcgga
     3121 gttccctatt ttgagctacg ccgatttcta ccagttggct ggcgttgttg ccgttgaggt
     3181 cacgggtgga cctaaggttc cattccaccc tggaagagag gacaagcctg agccaccacc
     3241 agagggtcgc ttgcccgatc ccactaaggg ttctgaccat ttgagagatg tgtttggcaa
     3301 agctatgggg cttactgacc aagatatcgt tgctctatct gggggtcaca ctattggagc
     3361 tgcacacaag gagcgttctg gatttgaggg tccctggacc tctaatcctc ttattttcga
     3421 caactcatac ttcacggagt tgttgagtgg tgagaaggaa ggtctccttc agctaccttc
     3481 tgacaaggct cttttgtctg accctgtatt ccgccctctc gttgacaaat atgcagcgga
     3541 cgaagatgcc ttctttgctg attacgctga ggctcaccaa aagctttccg agcttgggtt
     3601 tgctgatgcc ctgcagctgc ctcccctgga gcgcctgacc ctggactaag tcgagagatc
     3661 tatgaatcgt agatactgaa aaaccccgca agttcacttc aactgtgcat cgtgcaccat
     3721 ctcaatttct ttcatttata catcgttttg ccttctttta tgtaactata ctcctctaag
     3781 tttcaatctt ggccatgtaa cctctgatct atagaatttt ttaaatgact agaattaatg
     3841 cccatctttt ttttggacct aaattcttca tgaaaatata ttacgagggc ttattcagaa
     3901 gctttggact tcttcgccag aggtttggtc aagtctccaa tcaaggttgt cggcttgtct
     3961 accttgccag aaatttacga aaagatggaa aagggtcaaa tcgttggtag atacgttgtt
     4021 gacacttcta aataagcgaa tttcttatga tttatgattt ttattattaa ataagttata
     4081 aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg aaaattcttg
     4141 ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagca tgaggtcgct
     4201 cttattgacc acacctctac cggcatgccg gtcgagggac ctaataactt cgtatagcat
     4261 acattatacg aagttatatt aagggttatt gaatatgatc ggaattggtc gactacgtcg
     4321 ttaaggccgt ttctgacaga gtaaaattct tgagggaact ttcaccatta tgggaaatgg
     4381 ttcaagaagg tattgactta aactccatca aatggtcagg tcattgagtg ttttttattt
     4441 gttgtatttt ttttttttta gagaaaatcc tccaatatca aattaggaat cgtagtttca
     4501 tgattttctg ttacacctaa ctttttgtgt ggtgccctcc tccttgtcaa tattaatgtt
     4561 aaagtgcaat tctttttcct tatcacgttg agccattagt atcaatttgc ttacctgtat
     4621 tcctttacta tcctcctttt tctccttctt gataaatgta tgtagattgc gtatatagtt
     4681 tcgtctaccc tatgaacata ttccattttg taatttcgtg tcgtttctat tatgaatttc
     4741 atttataaag tttatgtaca aatatcataa aaaaagagaa tctttttaag caaggatttt
     4801 cttaacttct tcggcgacag catcaccgac ttcggtggta ctgttggaac cacctaaatc
     4861 accagttctg atacctgcat ccaaaacctt tttaactgca tcttcaatgg ccttaccttc
     4921 ttcaggcaag ttcaatgaca atttcaacat cattgcagca gacaagatag tggcgatagg
     4981 gtcaacctta ttctttggca aatctggagc agaaccgtgg catggttcgt acaaaccaaa
     5041 tgcggtgttc ttgtctggca aagaggccaa ggacgcagat ggcaacaaac ccaaggaacc
     5101 tgggataacg gaggcttcat cggagatgat atcaccaaac atgttgctgg tgattataat
     5161 accatttagg tgggttgggt tcttaactag gatcatggcg gcagaatcaa tcaattgatg
     5221 ttgaaccttc aatgtaggga attcgttctt gatggtttcc tccacagttt ttctccataa
     5281 tcttgaagag gccaaaacat tagctttatc caaggaccaa ataggcaatg gtggctcatg
     5341 ttgtagggcc atgaaagcgg ccattcttgt gattctttgc acttctggaa cggtgtattg
     5401 ttcactatcc caagcgacac catcaccatc gtcttccttt ctcttaccaa agtaaatacc
     5461 tcccactaat tctctgacaa caacgaagtc agtaccttta gcaaattgtg gcttgattgg
     5521 agataagtct aaaagagagt cggatgcaaa gttacatggt cttaagttgg cgtacaattg
     5581 aagttcttta cggattttta gtaaaccttg ttcaggtcta acactaccgg taccccattt
     5641 aggaccaccc acagcaccta acaaaacggc atcaaccttc ttggaggctt ccagcgcctc
     5701 atctggaagt gggacacctg tagcatcgat agcagcacca ccaattaaat gattttcgaa
     5761 atcgaacttg acattggaac gaacatcaga aatagcttta agaaccttaa tggcttcggc
     5821 tgtgatttct tgaccaacgt ggtcacctgg caaaacgacg atcttcttag gggcagacat
     5881 aggggcagac attagaatgg tatatccttg aaatatatat atatattgct gaaatgtaaa
     5941 aggtaagaaa agttagaaag taagacgatt gctaaccacc tattggaaaa aacaataggt
     6001 ccttaaataa tattgtcaac ttcaagtatt gtgatgcaag catttagtca tgaacgcttc
     6061 tctattctat atgaaaagcc ggttccggcc tctcaccttt cctttttctc ccaatttttc
     6121 agttgaaaaa ggtatatgcg tcaggcgacc tctgaaatta acaaaaaatt tccagtcatc
     6181 gaatttgatt ctgtgcgata gcgcccctgt gtgttctcgt tatgttgagg aaaaaaataa
     6241 tggttgctaa gagattcgaa ctcttgcatc ttacgatacc tgagtattcc cacagttcct
     6301 taccactctt ttgttactct attgatccag ctcagcaaag gcagtgtgat ctaagattct
     6361 atcttcgcga tgtagtaaaa ctagctagac cgagaaagag actagaaatg caaaaggcac
     6421 ttctacaatg gctgccatca ttattatccg atgtgacgct gcagcttctc aatgatattc
     6481 gaatacgctt tgaggagata cagcctaata tccgacaaac tgttttacag atttacgatc
     6541 gtacttgtta cccatcattg aattttgaac atccgaacct gggagttttc cctgaaacag
     6601 atagtatatt tgaacctgta taataatata tagtctagcg ctttacggaa gacaatgtat
     6661 gtatttcggt tcctggagaa actattgcat ctattgcata ggtaatcttg cacgtcgcat
     6721 ccccggttca ttttctgcgt ttccatcttg cacttcaata gcatatcttt gttaacgaag
     6781 catctgtgct tcattttgta gaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa
     6841 agaatctgag ctgcattttt acagaacaga aatgcaacgc gaaagcgcta ttttaccaac
     6901 gaagaatctg tgcttcattt ttgtaaaaca aaaatgcaac gcgagagcgc taatttttca
     6961 aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgagag cgctatttta
     7021 ccaacaaaga atctatactt cttttttgtt ctacaaaaat gcatcccgag agcgctattt
     7081 ttctaacaaa gcatcttaga ttactttttt tctcctttgt gcgctctata atgcagtctc
     7141 ttgataactt tttgcactgt aggtccgtta aggttagaag aaggctactt tggtgtctat
     7201 tttctcttcc ataaaaaaag cctgactcca cttcccgcgt ttactgatta ctagcgaagc
     7261 tgcgggtgca ttttttcaag ataaaggcat ccccgattat attctatacc gatgtggatt
     7321 gcgcatactt tgtgaacaga aagtgatagc gttgatgatt cttcattggt cagaaaatta
     7381 tgaacggttt cttctatttt gtctctatat actacgtata ggaaatgttt acattttcgt
     7441 attgttttcg attcactcta tgaatagttc ttactacaat ttttttgtct aaagagtaat
     7501 actagagata aacataaaaa atgtagaggt cgagtttaga tgcaagttca aggagcgaaa
     7561 ggtggatggg taggttatat agggatatag cacagagata tatagcaaag agatactttt
     7621 gagcaatgtt tgtggaagcg gtattcgcaa tattttagta gctcgttaca gtccggtgcg
     7681 tttttggttt tttgaaagtg cgtcttcaga gcgcttttgg ttttcaaaag cgctctgaag
     7741 ttcctatact ttctagagaa taggaacttc ggaataggaa cttcaaagcg tttccgaaaa
     7801 cgagcgcttc cgaaaatgca acgcgagctg cgcacataca gctcactgtt cacgtcgcac
     7861 ctatatctgc gtgttgcctg tatatatata tacatgagaa gaacggcata gtgcgtgttt
     7921 atgcttaaat gcgtacttat atgcgtctat ttatgtagga tgaaaggtag tctagtacct
     7981 cctgtgatat tatcccattc catgcggggt atcgtatgct tccttcagca ctacccttta
     8041 gctgttctat atgctgccac tcctcaattg gattagtctc atccttcaat gctatcattt
     8101 cctttgatat tggatcatat gcatagtacc gagaaactag tgcgaagtag tgatcaggta
     8161 ttgctgttat ctgatgagta tacgttgtcc tggccacggc agaagcacgc ttatcgctcc
     8221 aatttcccac aacattagtc aactccgtta ggcccttcat tgaaagaaat gaggtcatca
     8281 aatgtcttcc aatgtgagat tttgggccat tttttatagc aaagattgaa taaggcgcat
     8341 ttttcttcaa agc
//
