LOCUS       sequence-424259-        7859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing Tgl3-4V5-APEX2. Insufficient signal for EM. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7859)
  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 7859)
  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-p1k-Tgl3-4V5-APEX2-URA
FEATURES             Location/Qualifiers
     source          1..7859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2673..2714
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2721..2762
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2769..2810
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2817..2858
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     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      3686..3873
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3741..3765)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    3925..3958
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5410..5443
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5496..5515)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5497..5515)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5602..5619)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5773..6361)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5853..5872)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6532..7323,7324..7392))
                     /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     7155..7174
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7393..7497)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7565..7583
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7621..7643)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7743..7762
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7843..7859,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7843..7859,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg tgggattcct ggcactgttg
       61 tctcaaataa ttacttaaaa ctctagcatc tggagccaac ccggctaacg agagccccat
      121 gtgctcgtca catttgatga tttttttctg gtatgatgac aattcgtctg catttctttt
      181 caacgctact aaaacagcat gagtgttcga acgtaacccc acagtgacgc tcccttgttt
      241 aatagcttcc aaggcgtatt ccacttggaa cagacggcct gtaggagaaa aagtgactgt
      301 gtccccgtcg taattgttcc tgaacatgtt ttgtaaatat atgaaggctt agttcaatgt
      361 attcttaccc acttctcttg aacttcaatt gatcaccacg tctacctaaa tgtagtggcg
      421 tcttggttct tttccatact ttgacgatac ttttgaattt ttaattttgc cactctataa
      481 tagggtatgt tctttatagg tcatttttac ttttttagta gggcatttta ataaatacta
      541 ggtttttagt aatttcggag atacttatcc taggtctgaa aattcaaccc taaccctttt
      601 tttcttatgt tttttttttt ctgggtagca gctaatagaa attagtactg attacataga
      661 gaagtactat gacacaatag taagggaatc atctattcat atatcacatc tttgagttgc
      721 cgttaagcat gaaggaaacg gcgcaggaat acaaggtgtc tgctgtaata ccgacccttt
      781 tgaaaaactg gatactgcgt gtagtgtatg ccacattgga ccatatacct ccttttgtgt
      841 gggaaattct acatgtcatc accgatattt atttcttctg ggttcaaaag ctgattaact
      901 atgtgcggcc acactcaaga gtcatttatt ataacgctat taagaagtta gatgagtgtg
      961 atacgtatca aatgtggtgt cagcaagcgt ccgtagtgga tgaaataaca ggcgcaaatt
     1021 tatggcggcg gaatttcttt tcgaggaggt acgacttcaa ttctgtcatt gaacaatata
     1081 gcatattgga aaatatgtta agggaggaga agtatgatgt agttaaggag aagttttcga
     1141 ctacaggccc ttgtatgttg aggaactttg cgggtattgg agataaaaag ttgttcacaa
     1201 aatctttgat gggtacaaag ctacttattg aacaatattt gacccgaatt ctagaaggct
     1261 tggatatatt aaataatcaa actttaactc caacatcctt tttccaaaga tgtaagttgt
     1321 cactgggcac aacagcatta atcttgcaag gtggttcatt gtttgggctg tttcaccttg
     1381 gggtgattag aggtttattg ttacaggacc tgatgcctaa tattataagt ggtagctcta
     1441 tgggggcatg cgttgcaagt ttattcggtt gcttgtccaa tgagcaacta aagcaattat
     1501 tgaccgatga caacctcttg aatatcatca agaatgatgt agatttatta aaaagctgtg
     1561 gatacggtaa tttagagcag cacctaaacc tggggacttt gattcagaac ctaatacacc
     1621 acggttattc tcaagacgtt tatcttttta tccggtttgt tatgaaatat attgttaagg
     1681 aaaaaacttt tgaagaagtt tatcaaatta ctggaaaagt tttcaatatt gttattcatc
     1741 caactgataa atcatgccct aatttactaa attatgtgac aaccccaaac gtattaatca
     1801 agtcagccat tgaatgctca cttggttctg gtgtaatttc agaggataca tcactattat
     1861 gtaaaaattt agaaaatgaa attgagcctt ttcttaacat caataaaaat aaacaagtca
     1921 aatttcttac cccagaaaat gccaacaatc cgagcataac ggagagcccc tatactaggt
     1981 taacggaact attcaatgtt aataacttca ttgtttcttt ggcgagacca tacttggcac
     2041 cattggtggt aaatgatttg aagcatgaaa taaaaacgtc aaaatattac tattataagc
     2101 attatccaaa tatgcctcct ataaacgcga atacagtaag aaaaacacag agatcctctt
     2161 cgcaatcacc cataaaggca ggaactgtcg aggatcttga gccagagcct ttaatgagcc
     2221 ctgtgccccc aagttcggca gtcaacgatt cagcagagta cataatccca gaattgggca
     2281 taccgcagtt gaactttact gaaatggaac cgctggcgtt taaatttaag tatcatctgg
     2341 aaagaaaatt aaaaaatatt gccactatgg aatttcgaca taggatggag gtattggata
     2401 atttaggcct gttgtgctca ttaataaaaa gactaataat tgatgaaaag actcccagat
     2461 cagctacgga gattgctgtg gtaccaagga tgaaaagtct atcattaaca aggattattg
     2521 aaggtcaact aaacaacatt ccatactgga taaaatctgg tgaacgaagt acatggcccg
     2581 cactagcttt aattaagaca agatgtgcag tcgaatttaa attagacgac ataataagag
     2641 caagacggag taggttaatt aacatcgcta gcggtaaacc aattccaaat ccattgttgg
     2701 gtttggattc tactcctagc ggtaaaccaa ttccaaatcc attgttgggt ttggattcta
     2761 ctcctagcgg taaaccaatt ccaaatccat tgttgggttt ggattctact cctagcggta
     2821 aaccaattcc aaatccattg ttgggtttgg attctactcc taggggaaag 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 cctggacgct cagtgcagat
     3661 cttgaggcgc gccacttcta aataagcgaa tttcttatga tttatgattt ttattattaa
     3721 ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg
     3781 aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagta
     3841 tgaggtcgct cttattgacc acacctctac cggcagatcc gctagggata acagggtaat
     3901 atctgcaggt cgacaaccct taatataact tcgtataatg tatgctatac gaagttatta
     3961 ggtctagaga tcccaataca acagatcacg tgatcttttt gtaagatgaa gttgaagtag
     4021 tgttgcaccg tgccaatgca ggtggctatt agattaaata tgtgatttgt tctattaagt
     4081 ttcctgtata attaatgggg agcgctgatt ctcttttggt acgcttccca tccagcattt
     4141 ctgtatcttt caccttcaac cttaggatct ctacccttgg cgaaaagtcc tctgccaaca
     4201 atgatgatat ctgatccacc acttacaact tcgtcgacgg ttctgtactg ctgacccaat
     4261 gcatcgcctt tgtcgtctaa acctacacct ggggtcatga ttagccaatc aaacccttct
     4321 tctcttcctc ccatatcgtt ctgagcaatg aacccaataa cgaaatcttt atcactcttt
     4381 gcaatatcaa cggtaccctt agtatattca ccgtgtgcta gagaaccctt ggaagacaat
     4441 tcagcaagca tcaataatcc ccttggttct ttggtgacct cttgcgcacc ttgtttcaag
     4501 ccagcaacaa taccagcacc agtaaccccg tgggcgttgg tgatatcaga ccattctgcg
     4561 atacggtaaa cgcccgatgt atattgtaat ttgactgtgt taccgatatc ggcgaatttt
     4621 ctgtcctcaa atatcaagaa cttgtatttc tctgccaatg ctttcaatgg aacgacagta
     4681 ccctcataac tgaaatcatc caagatatca acgtgtgttt tcaaaaggca aatgtatgga
     4741 cccaacgttt caacaagttt caatagctca tcagtcgaac gaacgtcaag agaagcacac
     4801 aaattggtct tcttttcatc cattaaacgt aaaagtttcg atgcaaccgg acttgcatga
     4861 gtctcagctc tactggtata tgattttgtg gacatggtgc aactaattga cgggagtgta
     4921 ttgacgctgg cgtactggct ttcacaaaat ggcccaatca caaccacatc ttagatagtt
     4981 gaaatgactt tagataacat caattgagat gagcttaatc atgtcaaagc taaaagtgtc
     5041 accatgaacg acaattctta agcaaatcac gtgatataga tccacgaata accaccattt
     5101 gatgctcgag gcaagtaatg tgtgtaaaaa aatgcgttac caccatccaa tgcagaccga
     5161 tcttctaccc agaatcacat atatttatgt accgagtacc ttttttctat cttccaattg
     5221 cttctcccat atgattgtct ccgtaagctc gaaatttcta agttggattt taatcttcac
     5281 gcaggatgac agttcgatga gcttctgagg agtgtttaga acataatcat ttatccatgg
     5341 tctatctctt cttgtcgctt tttctcctcg atagaaccta aataaaacga gctctcgaga
     5401 acccttaata taacttcgta taatgtatgc tatacgaagt tattaggtga tatcagatcc
     5461 actagtggcc tatgcggccg cggatctgcc ggtctcccta tagtgagtcg tattaatttc
     5521 gataagccag gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5581 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5641 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5701 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5761 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5821 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5881 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5941 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6001 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6061 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6121 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6181 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6241 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6301 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6361 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6421 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6481 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6541 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6601 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6661 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6721 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6781 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6841 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6901 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6961 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7021 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7081 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7141 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7201 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7261 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7321 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7381 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7441 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7501 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     7561 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     7621 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     7681 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     7741 tgcggcatca gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg
     7801 cggctacaat taatacataa ccttatgtat catacacata cgatttaggt gacactata
//
