LOCUS       sequence-430371-        8212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing GFP-Pex3-4V5-APEX2 under ADH3 promoter. Insufficient 
            signal for EM..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8212)
  AUTHORS   Li H, He CW, Zhu J, Xie Z
  TITLE     A Validated Set of Ascorbate Peroxidase-Based Organelle Markers for 
            Electron Microscopy of Saccharomyces cerevisiae.
  JOURNAL   mSphere. 2022 Aug 31;7(4):e0010722. doi: 10.1128/msphere.00107-22. 
            Epub 2022 Jun 21.
  PUBMED    35727034
REFERENCE   2  (bases 1 to 8212)
  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: "mSphere."; 
            date: "2022-08-31"; volume: "7(4):e0010722. doi"; pages: " 
            10.1128/msphere.00107-22. Epub 2022 Jun 21"
COMMENT     Sequence Label: ClhN-pADH3-GFP-Pex3-4V5-APEX2-URA
FEATURES             Location/Qualifiers
     source          1..8212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      461..1049
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     950..969
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1203..1220
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1307..1326
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1307..1325
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(1379..1412)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(2864..2897)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      complement(2949..3136)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     3057..3081
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(3178..3204)
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             complement(3211..3957)
                     /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             complement(3964..4005)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(4012..4053)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(4060..4101)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(5437..6150)
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGIDELYK"
     promoter        complement(7173..7191)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(7174..7191)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(7272..7291)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7391..7413
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7451..7469)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7537..7641
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7642..7710,7711..8212,1..290)
                     /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     complement(7860..7879)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
       61 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
      121 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
      181 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
      241 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
      301 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
      361 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
      421 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
      481 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
      541 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
      601 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
      661 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
      721 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
      781 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
      841 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
      901 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
      961 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     1021 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     1081 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     1141 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     1201 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     1261 cgcgttggcc gattcattaa tgcaggttaa cctggcttat cgaaattaat acgactcact
     1321 atagggagac cggcagatcc gcggccgcat aggccactag tggatctgat atcacctaat
     1381 aacttcgtat agcatacatt atacgaagtt atattaaggg ttctcgagag ctcgttttat
     1441 ttaggttcta tcgaggagaa aaagcgacaa gaagagatag accatggata aatgattatg
     1501 ttctaaacac tcctcagaag ctcatcgaac tgtcatcctg cgtgaagatt aaaatccaac
     1561 ttagaaattt cgagcttacg gagacaatca tatgggagaa gcaattggaa gatagaaaaa
     1621 aggtactcgg tacataaata tatgtgattc tgggtagaag atcggtctgc attggatggt
     1681 ggtaacgcat ttttttacac acattacttg cctcgagcat caaatggtgg ttattcgtgg
     1741 atctatatca cgtgatttgc ttaagaattg tcgttcatgg tgacactttt agctttgaca
     1801 tgattaagct catctcaatt gatgttatct aaagtcattt caactatcta agatgtggtt
     1861 gtgattgggc cattttgtga aagccagtac gccagcgtca atacactccc gtcaattagt
     1921 tgcaccatgt ccacaaaatc atataccagt agagctgaga ctcatgcaag tccggttgca
     1981 tcgaaacttt tacgtttaat ggatgaaaag aagaccaatt tgtgtgcttc tcttgacgtt
     2041 cgttcgactg atgagctatt gaaacttgtt gaaacgttgg gtccatacat ttgccttttg
     2101 aaaacacacg ttgatatctt ggatgatttc agttatgagg gtactgtcgt tccattgaaa
     2161 gcattggcag agaaatacaa gttcttgata tttgaggaca gaaaattcgc cgatatcggt
     2221 aacacagtca aattacaata tacatcgggc gtttaccgta tcgcagaatg gtctgatatc
     2281 accaacgccc acggggttac tggtgctggt attgttgctg gcttgaaaca aggtgcgcaa
     2341 gaggtcacca aagaaccaag gggattattg atgcttgctg aattgtcttc caagggttct
     2401 ctagcacacg gtgaatatac taagggtacc gttgatattg caaagagtga taaagatttc
     2461 gttattgggt tcattgctca gaacgatatg ggaggaagag aagaagggtt tgattggcta
     2521 atcatgaccc caggtgtagg tttagacgac aaaggcgatg cattgggtca gcagtacaga
     2581 accgtcgacg aagttgtaag tggtggatca gatatcatca ttgttggcag aggacttttc
     2641 gccaagggta gagatcctaa ggttgaaggt gaaagataca gaaatgctgg atgggaagcg
     2701 taccaaaaga gaatcagcgc tccccattaa ttatacagga aacttaatag aacaaatcac
     2761 atatttaatc taatagccac ctgcattggc acggtgcaac actacttcaa cttcatctta
     2821 caaaaagatc acgtgatctg ttgtattggg atctctagac ctaataactt cgtatagcat
     2881 acattatacg aagttatatt aagggttgtc gacctgcaga tattaccctg ttatccctag
     2941 cggatctgcc ggtagaggtg tggtcaataa gagcgacctc atactatacc tgagaaagca
     3001 acctgaccta caggaaagag ttactcaaga ataagaattt tcgttttaaa acctaagagt
     3061 cactttaaaa tttgtataca cttatttttt ttataactta tttaataata aaaatcataa
     3121 atcataagaa attcgcttat ttagaagtgg cgcgcctcaa gatctgcact gagcgtccag
     3181 ggtcaggcgc tccaggggag gcagctgcag ggcatcagca aacccaagct cggaaagctt
     3241 ttggtgagcc tcagcgtaat cagcaaagaa ggcatcttcg tccgctgcat atttgtcaac
     3301 gagagggcgg aatacagggt cagacaaaag agccttgtca gaaggtagct gaaggagacc
     3361 ttccttctca ccactcaaca actccgtgaa gtatgagttg tcgaaaataa gaggattaga
     3421 ggtccaggga ccctcaaatc cagaacgctc cttgtgtgca gctccaatag tgtgaccccc
     3481 agatagagca acgatatctt ggtcagtaag ccccatagct ttgccaaaca catctctcaa
     3541 atggtcagaa cccttagtgg gatcgggcaa gcgaccctct ggtggtggct caggcttgtc
     3601 ctctcttcca gggtggaatg gaaccttagg tccacccgtg acctcaacgg caacaacgcc
     3661 agccaactgg tagaaatcgg cgtagctcaa aatagggaac tccgccttga gtggctccaa
     3721 aagcctaaca gcgatgtcaa gaccgttgtt agcgctgtga gccagttcgg cagggtgctt
     3781 gatggttccg aagggtccac cggtcttcgt gcccttgtca aaggttccag cagagtggaa
     3841 tgccaaacgg agcattagag gagcgcatct cttctcagcg atgaagcctc tgagcttctt
     3901 cttcgccttc tcaacggcgt cctggtaatc agcactcaca gttgggtaag actttcccct
     3961 aggagtagaa tccaaaccca acaatggatt tggaattggt ttaccgctag gagtagaatc
     4021 caaacccaac aatggatttg gaattggttt accgctagga gtagaatcca aacccaacaa
     4081 tggatttgga attggtttac cgctagcagg cttgaaggaa aacgagctgg agacgccaaa
     4141 gttgctgtat acgctggcgc tcagatcatc gagcggctgc acagaatcca gagtggccag
     4201 gtattcgttc acgctgccgc tatgggaaga cccggcagtt gtttgaagca tttcttggca
     4261 acagtctttc atcgacatag cgaaaactgc catttgatac tgttgctgat cttcttggcc
     4321 aggttttttc ttggctattg tctttatgcc gaccttgttc attatgaaat ggaatgattc
     4381 atttattaga gactctaaca ctatggatgt tgctgtgctt tcgatacatt gggtcagttc
     4441 attcaccaat tgattaaaaa ctagtgtgtt agagtgaaaa cttgttaaag catccgcgtc
     4501 aagcgtctgt gacagaagaa actcctgtcc actggaatct ttaggaagca aaatagacgt
     4561 gaggttatta ttattttgct ggaatatttg ggaattcgtg ttccggaata tgttcgtgag
     4621 acgagaacta aattcttcta acgttagagt atctcttggg tgtatgccgt caaattcaat
     4681 ttcgatttga tttgtaatta tttcattgta actcaaccac cccttgttaa gaatccacca
     4741 tgatagtgat aggaaagctt gctcattaat atacacttcc tggccttctt tctttgactt
     4801 ttttgctgcc attaccattg catcatccgc tttgtcctcc ggatcgctcc accacgatgt
     4861 aacccagtta tagaacctat tctgtagttt gttgcagttt tcctgctgca tggttaattt
     4921 tattgcggag tccaggtact cgtttcttgt caggatattt agttgcagtc ttgttaaaag
     4981 aatcagcgac gatactgtgt acgtcactgt caccagcttg atcaaactct ttagttctaa
     5041 ctcgttccac aactcagctt tactttttaa cggcgaactt tctctgcttt cactagactt
     5101 tgctctagtc aactggttct tttggtcctt taattgggta acgatgctgt ccaaattcaa
     5161 atcgttttca ttcaaaacca ttctccatac aggaagtagc tcgtatattg tgtacaacga
     5221 gtcttcctgt gtctgctcga accttctttt gatctgttct ttgatgaagt gttgctcagt
     5281 aatccgtaac tgctgtttat acaaccatct cttcacgaaa aacactacca ccgaccctgt
     5341 ggtaaataaa gcagctattc cagtcaatga aatgagtacc tttcctcgat gtctctgcag
     5401 aagcgaacgt gatctttgat ttggggccat cgtacgtttg tacaattcat caataccatg
     5461 ggtaatacca gcagcagtaa caaattctaa caagaccatg tggtctctct tttcgtttgg
     5521 atctttggat aaggcagatt gagtggataa gtaatggttg tctggtaaca agactggacc
     5581 atcaccaatt ggagtatttt gttgataatg gtcagctaat tgaacagaac catcttcaat
     5641 gttgtgtcta attttgaagt taactttgat accattcttt tgtttgtcag ccatgatgta
     5701 aacattgtga gagttatagt tgtattccaa tttgtgacct aaaatgttac catcttcttt
     5761 aaaatcaata ccttttaatt cgattctatt aactaaggta tcaccttcaa acttgacttc
     5821 agctctggtc ttgtagttac cgtcatcttt gaaaaaaata gttctttctt gaacataacc
     5881 ttctggcatg gcagacttga aaaagtcatg ttgtttcata tgatctgggt atctagaaaa
     5941 acattgaaca ccataagtta aagtagtgac taaggttggc catggaactg gcaatttacc
     6001 agtagtacaa ataaatttta aggtcaattt accgtaagta gcatcacctt caccttcacc
     6061 ggagacagaa aatttgtgac cattaacatc accatctaat tcaaccaaaa ttgggacaac
     6121 accagtgaat aattcttcac ctttagacat ccttttagtg gtttgcctcc ttgtttcgtg
     6181 cttctgcttt tacgatgact agtattaact acctcaataa tataccccat gctcatataa
     6241 gtcgaataac gcccttacca ttacgcacga ttaccaaaat gcaaggaatt tttaaggaaa
     6301 aagggaaata ttcatacatt cacgcaaact ctactacttg ttaacgacat cttagctatg
     6361 atcgctcaat agtgtcacta caaacgcata gactacgacg tgtggcaata ataacatacc
     6421 ttttatatgt cagagggaaa agtagacgaa tttatggcca tcactggcgc tgatgacgct
     6481 gctattgcca ctcaattcat cgaaatggca gacgggaact tgaacacagc catatcattg
     6541 tttttcgaaa atggtggggc tgctttactg agcagtaaca acacacctac tccgtctaat
     6601 tcgactccta tggcgcccac ctctgtagat tcagatgccg atgcacaatt agcggagagg
     6661 ttgcaaaggg aagcttacca acaacaacag cctgaccaag attatgtcag gccacctgat
     6721 gaggcaagac atgaagtact tactgaaact tcaggattcc caatttctta cggcggcatt
     6781 ggcggaagat ttgaaccatt gcatagggtg aacgacatgt ttgacgaagg aagacctgaa
     6841 agcattttta atcaacgatt ggatgacaca aatactaata catacatcaa tgacaactct
     6901 tcagattcct tggactccga ggaagaaaat gacgacgacg aatatgagta tgtggaagag
     6961 ccggtcatcg aacttgatga agacggcaat atcaaggagt acaccaaatt agtgagaaag
     7021 cctaaaacca tttccaagga gcagaaatta gctttgttat ttaggcctcc attttcaatc
     7081 atgtccaaac tcgacttaga cgctgcaaag caaaaggcaa gggcaaagca aaaatggata
     7141 atgattaaca aagcttcagc tggcggccgc gttctatagt gtcacctaaa tcgtatgtgt
     7201 atgatacata aggttatgta ttaattgtag ccgcgttcta acgacaatat gtccatatgg
     7261 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     7321 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     7381 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     7441 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     7501 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     7561 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     7621 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     7681 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     7741 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     7801 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     7861 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     7921 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     7981 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     8041 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     8101 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     8161 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa ac
//
