LOCUS       sequence-424573-        6943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Moderate overexpression of Snf7-4V5-APEX2 under ADH3 promoter. 
            Marker for late endosomes. Only partially functional. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6943)
  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 6943)
  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-Snf7-4V5-APEX2-URA
FEATURES             Location/Qualifiers
     source          1..6943
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1757..1798
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1805..1846
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1853..1894
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1901..1942
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1949..2695
                     /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             2702..2728
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      2770..2957
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2825..2849)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    3009..3042
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4494..4527
                     /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(4580..4599)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4581..4599)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4686..4703)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4857..5445)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4937..4956)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5616..6407,6408..6476))
                     /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     6239..6258
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6477..6581)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6649..6667
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6705..6727)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6827..6846
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6927..6943,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6927..6943,1)
                     /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 atgtggtcat cactttttgg ttggacatca agtaatgcca agaataaaga
     1081 gtcaccaaca aaggccatag tgcggttgag ggagcatatc aaccttctat ccaaaaagca
     1141 atcgcattta cgtactcaaa ttacaaatca agagaatgaa gctagaatct ttttgacgaa
     1201 gggcaataaa gtaatggcga agaatgcact taaaaagaag aagacaatcg aacaactttt
     1261 aagtaaggta gaaggcacaa tggagtctat ggaacagcag ctattctcta tagaaagtgc
     1321 aaacttaaat ctagagacaa tgagggctat gcaggaaggt gctaaggcaa tgaaaactat
     1381 tcacagtggc cttgacatag ataaagtgga tgaaactatg gacgagataa gggagcaagt
     1441 cgaattagga gatgaaataa gcgacgctat atccaggcct ttaattactg gggcaaacga
     1501 ggtggatgaa gatgagctgg acgaggaatt ggacatgctg gctcaagaaa atgctaacca
     1561 agaaacgtcc aagatcgtta ataataatgt taatgcggcg cctatctcag agaacaaagt
     1621 ctcactacct agtgttccaa gtaataaaat taaacaaagt gagaactctg tgaaggacgg
     1681 ggaagaggaa gaggatgaag aagatgaaga tgaaaaagca ttaagagaac tacaagcaga
     1741 aatggggctt gctagcggta aaccaattcc aaatccattg ttgggtttgg attctactcc
     1801 tagcggtaaa ccaattccaa atccattgtt gggtttggat tctactccta gcggtaaacc
     1861 aattccaaat ccattgttgg gtttggattc tactcctagc ggtaaaccaa ttccaaatcc
     1921 attgttgggt ttggattcta ctcctagggg aaagtcttac ccaactgtga gtgctgatta
     1981 ccaggacgcc gttgagaagg cgaagaagaa gctcagaggc ttcatcgctg agaagagatg
     2041 cgctcctcta atgctccgtt tggcattcca ctctgctgga acctttgaca agggcacgaa
     2101 gaccggtgga cccttcggaa ccatcaagca ccctgccgaa ctggctcaca gcgctaacaa
     2161 cggtcttgac atcgctgtta ggcttttgga gccactcaag gcggagttcc ctattttgag
     2221 ctacgccgat ttctaccagt tggctggcgt tgttgccgtt gaggtcacgg gtggacctaa
     2281 ggttccattc caccctggaa gagaggacaa gcctgagcca ccaccagagg gtcgcttgcc
     2341 cgatcccact aagggttctg accatttgag agatgtgttt ggcaaagcta tggggcttac
     2401 tgaccaagat atcgttgctc tatctggggg tcacactatt ggagctgcac acaaggagcg
     2461 ttctggattt gagggtccct ggacctctaa tcctcttatt ttcgacaact catacttcac
     2521 ggagttgttg agtggtgaga aggaaggtct ccttcagcta ccttctgaca aggctctttt
     2581 gtctgaccct gtattccgcc ctctcgttga caaatatgca gcggacgaag atgccttctt
     2641 tgctgattac gctgaggctc accaaaagct ttccgagctt gggtttgctg atgccctgca
     2701 gctgcctccc ctggagcgcc tgaccctgga cgctcagtgc agatcttgag gcgcgccact
     2761 tctaaataag cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaaa
     2821 ataagtgtat acaaatttta aagtgactct taggttttaa aacgaaaatt cttattcttg
     2881 agtaactctt tcctgtaggt caggttgctt tctcaggtat agtatgaggt cgctcttatt
     2941 gaccacacct ctaccggcag atccgctagg gataacaggg taatatctgc aggtcgacaa
     3001 cccttaatat aacttcgtat aatgtatgct atacgaagtt attaggtcta gagatcccaa
     3061 tacaacagat cacgtgatct ttttgtaaga tgaagttgaa gtagtgttgc accgtgccaa
     3121 tgcaggtggc tattagatta aatatgtgat ttgttctatt aagtttcctg tataattaat
     3181 ggggagcgct gattctcttt tggtacgctt cccatccagc atttctgtat ctttcacctt
     3241 caaccttagg atctctaccc ttggcgaaaa gtcctctgcc aacaatgatg atatctgatc
     3301 caccacttac aacttcgtcg acggttctgt actgctgacc caatgcatcg cctttgtcgt
     3361 ctaaacctac acctggggtc atgattagcc aatcaaaccc ttcttctctt cctcccatat
     3421 cgttctgagc aatgaaccca ataacgaaat ctttatcact ctttgcaata tcaacggtac
     3481 ccttagtata ttcaccgtgt gctagagaac ccttggaaga caattcagca agcatcaata
     3541 atccccttgg ttctttggtg acctcttgcg caccttgttt caagccagca acaataccag
     3601 caccagtaac cccgtgggcg ttggtgatat cagaccattc tgcgatacgg taaacgcccg
     3661 atgtatattg taatttgact gtgttaccga tatcggcgaa ttttctgtcc tcaaatatca
     3721 agaacttgta tttctctgcc aatgctttca atggaacgac agtaccctca taactgaaat
     3781 catccaagat atcaacgtgt gttttcaaaa ggcaaatgta tggacccaac gtttcaacaa
     3841 gtttcaatag ctcatcagtc gaacgaacgt caagagaagc acacaaattg gtcttctttt
     3901 catccattaa acgtaaaagt ttcgatgcaa ccggacttgc atgagtctca gctctactgg
     3961 tatatgattt tgtggacatg gtgcaactaa ttgacgggag tgtattgacg ctggcgtact
     4021 ggctttcaca aaatggccca atcacaacca catcttagat agttgaaatg actttagata
     4081 acatcaattg agatgagctt aatcatgtca aagctaaaag tgtcaccatg aacgacaatt
     4141 cttaagcaaa tcacgtgata tagatccacg aataaccacc atttgatgct cgaggcaagt
     4201 aatgtgtgta aaaaaatgcg ttaccaccat ccaatgcaga ccgatcttct acccagaatc
     4261 acatatattt atgtaccgag tacctttttt ctatcttcca attgcttctc ccatatgatt
     4321 gtctccgtaa gctcgaaatt tctaagttgg attttaatct tcacgcagga tgacagttcg
     4381 atgagcttct gaggagtgtt tagaacataa tcatttatcc atggtctatc tcttcttgtc
     4441 gctttttctc ctcgatagaa cctaaataaa acgagctctc gagaaccctt aatataactt
     4501 cgtataatgt atgctatacg aagttattag gtgatatcag atccactagt ggcctatgcg
     4561 gccgcggatc tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac
     4621 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4681 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4741 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4801 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4861 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4921 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4981 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5041 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5101 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5161 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5221 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5281 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5341 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5401 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5461 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5521 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5581 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5641 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5701 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5761 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5821 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5881 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5941 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6001 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6061 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6121 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6181 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6241 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6301 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6361 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6421 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6481 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6541 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6601 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     6661 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     6721 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     6781 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     6841 attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac
     6901 ataaccttat gtatcataca catacgattt aggtgacact ata
//
