LOCUS       sequence-199558-        8344 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 8344)
  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 8344)
  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..8344
                     /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..2817
                     /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     2788..2811
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             2841..2864
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2865..3611
                     /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             3618..3644
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      4037..4224
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4092..4116)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    complement(4245..4278)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(4787..5881)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(5882..6286)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      6763..8105
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(7726..7773)
                     /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 tcaaatgaac
     2581 gaatcaaatt aacaaccata ggatgataat gcgattagtt ttttagcctt atttctgggg
     2641 taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat gcaaaaactg
     2701 cataaccact ttaactaata ctttcaacat tttcggtttg tattacttct tattcaaatg
     2761 taataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt caaggagaaa
     2821 aaacaagctt tgcaaagatg gactacaagg atgacgacga taagggaaag tcttacccaa
     2881 ctgtgagtgc tgattaccag gacgccgttg agaaggcgaa gaagaagctc agaggcttca
     2941 tcgctgagaa gagatgcgct cctctaatgc tccgtttggc attccactct gctggaacct
     3001 ttgacaaggg cacgaagacc ggtggaccct tcggaaccat caagcaccct gccgaactgg
     3061 ctcacagcgc taacaacggt cttgacatcg ctgttaggct tttggagcca ctcaaggcgg
     3121 agttccctat tttgagctac gccgatttct accagttggc tggcgttgtt gccgttgagg
     3181 tcacgggtgg acctaaggtt ccattccacc ctggaagaga ggacaagcct gagccaccac
     3241 cagagggtcg cttgcccgat cccactaagg gttctgacca tttgagagat gtgtttggca
     3301 aagctatggg gcttactgac caagatatcg ttgctctatc tgggggtcac actattggag
     3361 ctgcacacaa ggagcgttct ggatttgagg gtccctggac ctctaatcct cttattttcg
     3421 acaactcata cttcacggag ttgttgagtg gtgagaagga aggtctcctt cagctacctt
     3481 ctgacaaggc tcttttgtct gaccctgtat tccgccctct cgttgacaaa tatgcagcgg
     3541 acgaagatgc cttctttgct gattacgctg aggctcacca aaagctttcc gagcttgggt
     3601 ttgctgatgc cctgcagctg cctcccctgg agcgcctgac cctggactaa gtcgagagat
     3661 ctatgaatcg tagatactga aaaaccccgc aagttcactt caactgtgca tcgtgcacca
     3721 tctcaatttc tttcatttat acatcgtttt gccttctttt atgtaactat actcctctaa
     3781 gtttcaatct tggccatgta acctctgatc tatagaattt tttaaatgac tagaattaat
     3841 gcccatcttt tttttggacc taaattcttc atgaaaatat attacgaggg cttattcaga
     3901 agctttggac ttcttcgcca gaggtttggt caagtctcca atcaaggttg tcggcttgtc
     3961 taccttgcca gaaatttacg aaaagatgga aaagggtcaa atcgttggta gatacgttgt
     4021 tgacacttct aaataagcga atttcttatg atttatgatt tttattatta aataagttat
     4081 aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt
     4141 attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc
     4201 tcttattgac cacacctcta ccggcatgcc ggtcgaggga cctaataact tcgtatagca
     4261 tacattatac gaagttatat taagggttat tgaatatgat cggaattggt cgactacgtc
     4321 gttaaggccg tttctgacag agtaaaattc ttgagggaac tttcaccatt atgggaaatg
     4381 gttcaagaag gtattgactt aaactccatc aaatggtcag gtcattgagt gttttttatt
     4441 tgttgtattt tttttttttt agagaaaatc ctccaatata taaattagga atcatagttt
     4501 catgattttc tgttacacct aactttttgt gtggtgccct cctccttgtc aatattaatg
     4561 ttaaagtgca attctttttc cttatcacgt tgagccatta gtatcaattt gcttacctgt
     4621 attcctttac atcctccttt ttctccttct tgataaatgt atgtagattg cgtatatagt
     4681 ttcgtctacc ctatgaacat attccatttt gtaatttcgt gtcgtttcta ttatgaattt
     4741 catttataaa gtttatgtac aaatatcata aaaaaagaga atctttttaa gcaaggattt
     4801 tcttaacttc ttcggcgaca gcatcaccga cttcggtggt actgttggaa ccacctaaat
     4861 caccagttct gatacctgca tccaaaacct ttttaactgc atcttcaatg gccttacctt
     4921 cttcaggcaa gttcaatgac aatttcaaca tcattgcagc agacaagata gtggcgatag
     4981 ggttgacctt attctttggc aaatctggag cagaaccgtg gcatggttcg tacaaaccaa
     5041 atgcggtgtt cttgtctggc aaagaggcca aggacgcaga tggcaacaaa cccaaggaac
     5101 ctgggataac ggaggcttca tcggagatga tatcaccaaa catgttgctg gtgattataa
     5161 taccatttag gtgggttggg ttcttaacta ggatcatggc ggcagaatca atcaattgat
     5221 gttgaacctt caatgtaggg aattcgttct tgatggtttc ctccacagtt tttctccata
     5281 atcttgaaga ggccaaaaca ttagctttat ccaaggacca aataggcaat ggtggctcat
     5341 gttgtagggc catgaaagcg gccattcttg tgattctttg cacttctgga acggtgtatt
     5401 gttcactatc ccaagcgaca ccatcaccat cgtcttcctt tctcttacca aagtaaatac
     5461 ctcccactaa ttctctgaca acaacgaagt cagtaccttt agcaaattgt ggcttgattg
     5521 gagataagtc taaaagagag tcggatgcaa agttacatgg tcttaagttg gcgtacaatt
     5581 gaagttcttt acggattttt agtaaacctt gttcaggtct aacactaccg gtaccccatt
     5641 taggaccacc cacagcacct aacaaaacgg catcagcctt cttggaggct tccagcgcct
     5701 catctggaag tggaacacct gtagcatcga tagcagcacc accaattaaa tgattttcga
     5761 aatcgaactt gacattggaa cgaacatcag aaatagcttt aagaacctta atggcttcgg
     5821 ctgtgatttc ttgaccaacg tggtcacctg gcaaaacgac gatcttctta ggggcagaca
     5881 ttagaatggt atatccttga aatatatata tatattgctg aaatgtaaaa ggtaagaaaa
     5941 gttagaaagt aagacgattg ctaaccacct attggaaaaa acaataggtc cttaaataat
     6001 attgtcaact tcaagtattg tgatgcaagc atttagtcat gaacgcttct ctattctata
     6061 tgaaaagccg gttccggcgc tctcaccttt cctttttctc ccaatttttc agttgaaaaa
     6121 ggtatatgcg tcaggcgacc tctgaaatta acaaaaaatt tccagtcatc gaatttgatt
     6181 ctgtgcgata gcgcccctgt gtgttctcgt tatgttgagg aaaaaaataa tggttgctaa
     6241 gagattcgaa ctcttgcatc ttacgatact gagtattccc acagttcctt accactcttt
     6301 tgttactcta ttgatccagc tcagcaaagg cagtgtgatc taagattcta tcttcgcgat
     6361 gtagtaaaac tagctagacc gagaaagaga ctagaaatgc aaaaggcact tctacaatgg
     6421 ctgccatcat tattatccga tgtgacgctg cagcttctca atgatattcg aatacgcttt
     6481 gaggagatac agcctaatat ccgacaaact gttttacaga tttacgatcg tacttgttac
     6541 ccatcattga attttgaaca tccgaacctg ggagttttcc ctgaaacaga tagtatattt
     6601 gaacctgtat aataatatat agtctagcgc tttacggaag acaatgtatg tatttcggtt
     6661 cctggagaaa ctattgcatc tattgcatag gtaatcttgc acgtcgcatc cccggttcat
     6721 tttctgcgtt tccatcttgc acttcaatag catatctttg ttaacgaagc atctgtgctt
     6781 cattttgtag aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc
     6841 tgcattttta cagaacagaa atgcaacgcg aaagcgctat tttaccaacg aagaatctgt
     6901 gcttcatttt tgtaaaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc
     6961 tgagctgcat ttttacagaa cagaaatgca acgcgagagc gctattttac caacaaagaa
     7021 tctatacttc ttttttgttc tacaaaaatg catcccgaga gcgctatttt tctaacaaag
     7081 catcttagat tacttttttt ctcctttgtg cgctctataa tgcagtctct tgataacttt
     7141 ttgcactgta ggtccgttaa ggttagaaga aggctacttt ggtgtctatt ttctcttcca
     7201 taaaaaaagc ctgactccac ttcccgcgtt tactgattac tagcgaagct gcgggtgcat
     7261 tttttcaaga taaaggcatc cccgattata ttctataccg atgtggattg cgcatacttt
     7321 gtgaacagaa agtgatagcg ttgatgattc ttcattggtc agaaaattat gaacggtttc
     7381 ttctattttg tctctatata ctacgtatag gaaatgttta cattttcgta ttgttttcga
     7441 ttcactctat gaatagttct tactacaatt tttttgtcta aagagtaata ctagagataa
     7501 acataaaaaa tgtagaggtc gagtttagat gcaagttcaa ggagcgaaag gtggatgggt
     7561 aggttatata gggatatagc acagagatat atagcaaaga gatacttttg agcaatgttt
     7621 gtggaagcgg tattcgcaat attttagtag ctcgttacag tccggtgcgt ttttggtttt
     7681 ttgaaagtgc gtcttcagag cgcttttggt tttcaaaagc gctctgaagt tcctatactt
     7741 tctagagaat aggaacttcg gaataggaac ttcaaagcgt ttccgaaaac gagcgcttcc
     7801 gaaaatgcaa cgcgagctgc gcacatacag ctcactgttc acgtcgcacc tatatctgcg
     7861 tgttgcctgt atatatatat acatgagaag aacggcatag tgcgtgttta tgcttaaatg
     7921 cgtacttata tgcgtctatt tatgtaggat gaaaggtagt ctagtacctc ctgtgatatt
     7981 atcccattcc atgcggggta tcgtatgctt ccttcagcac taccctttag ctgttctata
     8041 tgctgccact cctcaattgg attagtctca tccttcaatg ctatcatttc ctttgatatt
     8101 ggatcatatg catagtaccg agaaactagt gcgaagtagt gatcaggtat tgctgttatc
     8161 tgatgagtat acgttgtcct ggccacggca gaagcacgct tatcgctcca atttcccaca
     8221 acattagtca actccgttag gcccttcatt gaaagaaatg aggtcatcaa atgtcttcca
     8281 atgtgagatt ttgggccatt ttttatagca aagattgaat aaggcgcatt tttcttcaaa
     8341 gctc
//
