LOCUS       sequence-424571-        8149 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing Tgl3-4V5-APEX2. Marker for lipid droplets..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8149)
  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 8149)
  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-Tgl3-4V5-APEX2-URA
FEATURES             Location/Qualifiers
     source          1..8149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2963..3004
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3011..3052
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3059..3100
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3107..3148
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3155..3901
                     /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             3908..3934
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      3976..4163
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4031..4055)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    4215..4248
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5700..5733
                     /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(5786..5805)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5787..5805)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5892..5909)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6063..6651)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6143..6162)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6822..7613,7614..7682))
                     /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     7445..7464
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7683..7787)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7855..7873
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7911..7933)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8033..8052
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(8133..8149,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(8133..8149,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tccttacgaa aaaaaagatc aaggaaacat
       61 gtcggaaaca aatgggaaac aacaaaaagg acttatcatt cctacttttt tgtcgagggc
      121 atttgtcgag ggtaatactg catactgcct attgtcggtc ccgcagaatt aatagtggct
      181 gctcatacac aacgaaaaaa attgttaaca aaatagaagg attcgtctat gccctttttt
      241 cagtgcggaa attatatacc acacaagccg tcgaagccca tagggtcctt taacaataaa
      301 gactctccta aaaaaatttt tcttgtgaca cgtcagtgca ggatataaat gtagcaagcg
      361 tatataacaa atgacaaagg gttcttgcgc aaggttactt tagcgttgct ggataatgat
      421 cggattgttt ttagatcacg atagcacatt tgaggaatcg tatcgaaacg gacagggcaa
      481 gagtacgagg gcgaaatgaa cttataacac cattgatatt ttctcctcaa tcttatcatt
      541 ccacgcacat tattttccta ctatgttatg cgggaacagt tcctaaggaa tttttctaat
      601 ggcatttcgg cagcgtcatc aaatgtacgc acccacttca agtgatcatt cgctcgttac
      661 tacctatcac gtgtcgcatc actcgcgacg tccatgcgca tgctgtgccg caggagaatt
      721 cgagatgcga atgagcagca gccattttga tgttgtgagc atcggaacgt ttctgcgtcc
      781 gtacactgtc cttttgttac ttagataatg gctaaggcaa gcagtccggg ccacaggagt
      841 caaaggcttt tcgccagctc ctaaacgctg gaagtgtaat tttttttcgt cttataaaat
      901 taaacaaacc cttttagaaa ggaacactcg ctttatctct tcgaccgaat ttactataca
      961 tggatatata tatattatct tctgttcaca gttaaaacta ggaatagtat agtcataagt
     1021 taacaccatc atgaaggaaa cggcgcagga atacaaggtg tctgctgtaa taccgaccct
     1081 tttgaaaaac tggatactgc gtgtagtgta tgccacattg gaccatatac ctccttttgt
     1141 gtgggaaatt ctacatgtca tcaccgatat ttatttcttc tgggttcaaa agctgattaa
     1201 ctatgtgcgg ccacactcaa gagtcattta ttataacgct attaagaagt tagatgagtg
     1261 tgatacgtat caaatgtggt gtcagcaagc gtccgtagtg gatgaaataa caggcgcaaa
     1321 tttatggcgg cggaatttct tttcgaggag gtacgacttc aattctgtca ttgaacaata
     1381 tagcatattg gaaaatatgt taagggagga gaagtatgat gtagttaagg agaagttttc
     1441 gactacaggc ccttgtatgt tgaggaactt tgcgggtatt ggagataaaa agttgttcac
     1501 aaaatctttg atgggtacaa agctacttat tgaacaatat ttgacccgaa ttctagaagg
     1561 cttggatata ttaaataatc aaactttaac tccaacatcc tttttccaaa gatgtaagtt
     1621 gtcactgggc acaacagcat taatcttgca aggtggttca ttgtttgggc tgtttcacct
     1681 tggggtgatt agaggtttat tgttacagga cctgatgcct aatattataa gtggtagctc
     1741 tatgggggca tgcgttgcaa gtttattcgg ttgcttgtcc aatgagcaac taaagcaatt
     1801 attgaccgat gacaacctct tgaatatcat caagaatgat gtagatttat taaaaagctg
     1861 tggatacggt aatttagagc agcacctaaa cctggggact ttgattcaga acctaataca
     1921 ccacggttat tctcaagacg tttatctttt tatccggttt gttatgaaat atattgttaa
     1981 ggaaaaaact tttgaagaag tttatcaaat tactggaaaa gttttcaata ttgttattca
     2041 tccaactgat aaatcatgcc ctaatttact aaattatgtg acaaccccaa acgtattaat
     2101 caagtcagcc attgaatgct cacttggttc tggtgtaatt tcagaggata catcactatt
     2161 atgtaaaaat ttagaaaatg aaattgagcc ttttcttaac atcaataaaa ataaacaagt
     2221 caaatttctt accccagaaa atgccaacaa tccgagcata acggagagcc cctatactag
     2281 gttaacggaa ctattcaatg ttaataactt cattgtttct ttggcgagac catacttggc
     2341 accattggtg gtaaatgatt tgaagcatga aataaaaacg tcaaaatatt actattataa
     2401 gcattatcca aatatgcctc ctataaacgc gaatacagta agaaaaacac agagatcctc
     2461 ttcgcaatca cccataaagg caggaactgt cgaggatctt gagccagagc ctttaatgag
     2521 ccctgtgccc ccaagttcgg cagtcaacga ttcagcagag tacataatcc cagaattggg
     2581 cataccgcag ttgaacttta ctgaaatgga accgctggcg tttaaattta agtatcatct
     2641 ggaaagaaaa ttaaaaaata ttgccactat ggaatttcga cataggatgg aggtattgga
     2701 taatttaggc ctgttgtgct cattaataaa aagactaata attgatgaaa agactcccag
     2761 atcagctacg gagattgctg tggtaccaag gatgaaaagt ctatcattaa caaggattat
     2821 tgaaggtcaa ctaaacaaca ttccatactg gataaaatct ggtgaacgaa gtacatggcc
     2881 cgcactagct ttaattaaga caagatgtgc agtcgaattt aaattagacg acataataag
     2941 agcaagacgg agtagggcta gcggtaaacc aattccaaat ccattgttgg gtttggattc
     3001 tactcctagc ggtaaaccaa ttccaaatcc attgttgggt ttggattcta ctcctagcgg
     3061 taaaccaatt ccaaatccat tgttgggttt ggattctact cctagcggta aaccaattcc
     3121 aaatccattg ttgggtttgg attctactcc taggggaaag tcttacccaa ctgtgagtgc
     3181 tgattaccag gacgccgttg agaaggcgaa gaagaagctc agaggcttca tcgctgagaa
     3241 gagatgcgct cctctaatgc tccgtttggc attccactct gctggaacct ttgacaaggg
     3301 cacgaagacc ggtggaccct tcggaaccat caagcaccct gccgaactgg ctcacagcgc
     3361 taacaacggt cttgacatcg ctgttaggct tttggagcca ctcaaggcgg agttccctat
     3421 tttgagctac gccgatttct accagttggc tggcgttgtt gccgttgagg tcacgggtgg
     3481 acctaaggtt ccattccacc ctggaagaga ggacaagcct gagccaccac cagagggtcg
     3541 cttgcccgat cccactaagg gttctgacca tttgagagat gtgtttggca aagctatggg
     3601 gcttactgac caagatatcg ttgctctatc tgggggtcac actattggag ctgcacacaa
     3661 ggagcgttct ggatttgagg gtccctggac ctctaatcct cttattttcg acaactcata
     3721 cttcacggag ttgttgagtg gtgagaagga aggtctcctt cagctacctt ctgacaaggc
     3781 tcttttgtct gaccctgtat tccgccctct cgttgacaaa tatgcagcgg acgaagatgc
     3841 cttctttgct gattacgctg aggctcacca aaagctttcc gagcttgggt ttgctgatgc
     3901 cctgcagctg cctcccctgg agcgcctgac cctggacgct cagtgcagat cttgaggcgc
     3961 gccacttcta aataagcgaa tttcttatga tttatgattt ttattattaa ataagttata
     4021 aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg aaaattctta
     4081 ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagta tgaggtcgct
     4141 cttattgacc acacctctac cggcagatcc gctagggata acagggtaat atctgcaggt
     4201 cgacaaccct taatataact tcgtataatg tatgctatac gaagttatta ggtctagaga
     4261 tcccaataca acagatcacg tgatcttttt gtaagatgaa gttgaagtag tgttgcaccg
     4321 tgccaatgca ggtggctatt agattaaata tgtgatttgt tctattaagt ttcctgtata
     4381 attaatgggg agcgctgatt ctcttttggt acgcttccca tccagcattt ctgtatcttt
     4441 caccttcaac cttaggatct ctacccttgg cgaaaagtcc tctgccaaca atgatgatat
     4501 ctgatccacc acttacaact tcgtcgacgg ttctgtactg ctgacccaat gcatcgcctt
     4561 tgtcgtctaa acctacacct ggggtcatga ttagccaatc aaacccttct tctcttcctc
     4621 ccatatcgtt ctgagcaatg aacccaataa cgaaatcttt atcactcttt gcaatatcaa
     4681 cggtaccctt agtatattca ccgtgtgcta gagaaccctt ggaagacaat tcagcaagca
     4741 tcaataatcc ccttggttct ttggtgacct cttgcgcacc ttgtttcaag ccagcaacaa
     4801 taccagcacc agtaaccccg tgggcgttgg tgatatcaga ccattctgcg atacggtaaa
     4861 cgcccgatgt atattgtaat ttgactgtgt taccgatatc ggcgaatttt ctgtcctcaa
     4921 atatcaagaa cttgtatttc tctgccaatg ctttcaatgg aacgacagta ccctcataac
     4981 tgaaatcatc caagatatca acgtgtgttt tcaaaaggca aatgtatgga cccaacgttt
     5041 caacaagttt caatagctca tcagtcgaac gaacgtcaag agaagcacac aaattggtct
     5101 tcttttcatc cattaaacgt aaaagtttcg atgcaaccgg acttgcatga gtctcagctc
     5161 tactggtata tgattttgtg gacatggtgc aactaattga cgggagtgta ttgacgctgg
     5221 cgtactggct ttcacaaaat ggcccaatca caaccacatc ttagatagtt gaaatgactt
     5281 tagataacat caattgagat gagcttaatc atgtcaaagc taaaagtgtc accatgaacg
     5341 acaattctta agcaaatcac gtgatataga tccacgaata accaccattt gatgctcgag
     5401 gcaagtaatg tgtgtaaaaa aatgcgttac caccatccaa tgcagaccga tcttctaccc
     5461 agaatcacat atatttatgt accgagtacc ttttttctat cttccaattg cttctcccat
     5521 atgattgtct ccgtaagctc gaaatttcta agttggattt taatcttcac gcaggatgac
     5581 agttcgatga gcttctgagg agtgtttaga acataatcat ttatccatgg tctatctctt
     5641 cttgtcgctt tttctcctcg atagaaccta aataaaacga gctctcgaga acccttaata
     5701 taacttcgta taatgtatgc tatacgaagt tattaggtga tatcagatcc actagtggcc
     5761 tatgcggccg cggatctgcc ggtctcccta tagtgagtcg tattaatttc gataagccag
     5821 gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5881 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5941 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     6001 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6061 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6121 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6181 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6241 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6301 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6361 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6421 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6481 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     6541 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6601 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6661 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6721 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6781 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6841 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6901 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6961 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     7021 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7081 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     7141 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7201 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     7261 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     7321 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     7381 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     7441 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     7501 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     7561 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7621 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7681 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7741 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7801 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     7861 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     7921 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     7981 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     8041 gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat
     8101 taatacataa ccttatgtat catacacata cgatttaggt gacactata
//
