LOCUS       sequence-422538-        7090 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing Vrg4-V5-APEX2. Marker for late Golgi/early endosomes. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7090)
  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 7090)
  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-Vrg4-V5-APEX2-URA
FEATURES             Location/Qualifiers
     source          1..7090
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2048..2089
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2096..2842
                     /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             2849..2875
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     terminator      2917..3104
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2972..2996)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    3156..3189
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4641..4674
                     /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(4727..4746)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4728..4746)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4833..4850)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5004..5592)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5084..5103)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5763..6554,6555..6623))
                     /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     6386..6405
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6624..6728)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6796..6814
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6852..6874)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6974..6993
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7074..7090,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7074..7090,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttgatctc aaggaaaaaa atgcaatgtc gggcgccaat
       61 acttatgatc atgcttagtc tcgacattga ccttgcttgc tacagtaatc tggtgtccga
      121 gtagtgacat ctaggtaccg gtgagaacta tatcttcatt actacacttg tcgcccctga
      181 atattatcgc tacacacgcg aaagagctct tgttaaagtt actctcgcat cattgcggta
      241 catgttttgc atattcatct gaagtaggaa agggtataag cagtatatga tgtaccatct
      301 gcataagcgg tgtttaatgc gtacctatcg tttaagaaca aaaggaacac acccacaccc
      361 gctccaatac catattgaga gaataatgag gtaacatatg cttctatttg gttcgataag
      421 acggctacta gtccaaaaaa agtcaaacaa caatttcaac agctacattc tgaagataga
      481 acgcattttt gcctgaatat tgcaactcaa tagacattct aaagtgctac gagaaaaaag
      541 cttgtaatgg accatggcaa tgtgcgccat atatatatgc ggctccctta tgagtagaag
      601 aaaatatatg actggtggta caaagtggca tggtggtata tagatgtagg aacaccattt
      661 gagtttgcca taatgattaa atttcccttt ttataaagaa ttagccgccc ataacacgaa
      721 aaatatcaca gttgacgaaa gaagacacgt cgcctaaacg cgtaatgaat cgcaatatac
      781 gaaaagctaa acgggttttt cagaatcctc ttacccagcc ttggataata gttttggccg
      841 aaaaaaaaaa aaagaaagaa aaaaattgaa atttcgcgat ccgaacaaac aatgaacaga
      901 aaaacagaat atatataacg ctgccaagcc tatttcttgt tgccgattaa ttgttgacat
      961 cctcttaatt accaaaagag cctaagaaaa caaacacact aaccacacag tatctttcgc
     1021 ccgacgtacg atgtctgaat tgaaaacagg tcatgcaggc cataaccctt gggcttcagt
     1081 tgccaattcc ggtccgatct ctattttatc ctactgtggt tcctctattt taatgacggt
     1141 gactaacaag ttcgtcgtca atttgaagga tttcaacatg aactttgtca tgcttttcgt
     1201 gcaatctttg gtttgtacta taaccttgat tatcctacgt atactgggct atgcgaagtt
     1261 ccgttcatta aacaaaacag acgccaagaa ctggttccct atttcctttt tactggtctt
     1321 gatgatctac acctcttcga aggctttaca atacttggct gttccaattt acaccatttt
     1381 caagaatttg actattatct tgattgctta tggtgaggtt ctcttttttg gtggctctgt
     1441 cacctccatg gaattgtcat catttttgtt gatggtcctt tcttctgtcg ttgcaacttg
     1501 gggtgaccag caagctgtgg ctgccaaggc tgcttcattg gctgaaggag cagccggtgc
     1561 tgttgcctcc tttaacccag gttatttctg gatgttcacc aactgtatca cttctgcatt
     1621 attcgttctt ataatgagaa agagaattaa gttaactaac ttcaaggatt tcgacactat
     1681 gttttacaac aatgttttgg ctctacctat tctattgctg ttttctttct gtgtggaaga
     1741 ttggtcttca gttaatttga ccaataactt ttctaacgat tcgctaactg ctatgatcat
     1801 cagtggtgtt gcatccgtcg gtatttctta ctgttccggt tggtgtgttc gtgttacttc
     1861 gtctactaca tattcgatgg taggggcttt gaacaagctg ccaattgcct tgtctggttt
     1921 gattttcttt gatgctccaa gaaacttctt atctattctc tccattttta ttggtttcct
     1981 atcaggtatt atttatgctg ttgccaaaca aaagaagcaa caagcccaac ctttacgtaa
     2041 agctagcggt aaaccaattc caaatccatt gttgggtttg gattctactc ctaggggaaa
     2101 gtcttaccca actgtgagtg ctgattacca ggacgccgtt gagaaggcga agaagaagct
     2161 cagaggcttc atcgctgaga agagatgcgc tcctctaatg ctccgtttgg cattccactc
     2221 tgctggaacc tttgacaagg gcacgaagac cggtggaccc ttcggaacca tcaagcaccc
     2281 tgccgaactg gctcacagcg ctaacaacgg tcttgacatc gctgttaggc ttttggagcc
     2341 actcaaggcg gagttcccta ttttgagcta cgccgatttc taccagttgg ctggcgttgt
     2401 tgccgttgag gtcacgggtg gacctaaggt tccattccac cctggaagag aggacaagcc
     2461 tgagccacca ccagagggtc gcttgcccga tcccactaag ggttctgacc atttgagaga
     2521 tgtgtttggc aaagctatgg ggcttactga ccaagatatc gttgctctat ctgggggtca
     2581 cactattgga gctgcacaca aggagcgttc tggatttgag ggtccctgga cctctaatcc
     2641 tcttattttc gacaactcat acttcacgga gttgttgagt ggtgagaagg aaggtctcct
     2701 tcagctacct tctgacaagg ctcttttgtc tgaccctgta ttccgccctc tcgttgacaa
     2761 atatgcagcg gacgaagatg ccttctttgc tgattacgct gaggctcacc aaaagctttc
     2821 cgagcttggg tttgctgatg ccctgcagct gcctcccctg gagcgcctga ccctggacgc
     2881 tcagtgcaga tcttgaggcg cgccacttct aaataagcga atttcttatg atttatgatt
     2941 tttattatta aataagttat aaaaaaaata agtgtataca aattttaaag tgactcttag
     3001 gttttaaaac gaaaattctt attcttgagt aactctttcc tgtaggtcag gttgctttct
     3061 caggtatagt atgaggtcgc tcttattgac cacacctcta ccggcagatc cgctagggat
     3121 aacagggtaa tatctgcagg tcgacaaccc ttaatataac ttcgtataat gtatgctata
     3181 cgaagttatt aggtctagag atcccaatac aacagatcac gtgatctttt tgtaagatga
     3241 agttgaagta gtgttgcacc gtgccaatgc aggtggctat tagattaaat atgtgatttg
     3301 ttctattaag tttcctgtat aattaatggg gagcgctgat tctcttttgg tacgcttccc
     3361 atccagcatt tctgtatctt tcaccttcaa ccttaggatc tctacccttg gcgaaaagtc
     3421 ctctgccaac aatgatgata tctgatccac cacttacaac ttcgtcgacg gttctgtact
     3481 gctgacccaa tgcatcgcct ttgtcgtcta aacctacacc tggggtcatg attagccaat
     3541 caaacccttc ttctcttcct cccatatcgt tctgagcaat gaacccaata acgaaatctt
     3601 tatcactctt tgcaatatca acggtaccct tagtatattc accgtgtgct agagaaccct
     3661 tggaagacaa ttcagcaagc atcaataatc cccttggttc tttggtgacc tcttgcgcac
     3721 cttgtttcaa gccagcaaca ataccagcac cagtaacccc gtgggcgttg gtgatatcag
     3781 accattctgc gatacggtaa acgcccgatg tatattgtaa tttgactgtg ttaccgatat
     3841 cggcgaattt tctgtcctca aatatcaaga acttgtattt ctctgccaat gctttcaatg
     3901 gaacgacagt accctcataa ctgaaatcat ccaagatatc aacgtgtgtt ttcaaaaggc
     3961 aaatgtatgg acccaacgtt tcaacaagtt tcaatagctc atcagtcgaa cgaacgtcaa
     4021 gagaagcaca caaattggtc ttcttttcat ccattaaacg taaaagtttc gatgcaaccg
     4081 gacttgcatg agtctcagct ctactggtat atgattttgt ggacatggtg caactaattg
     4141 acgggagtgt attgacgctg gcgtactggc tttcacaaaa tggcccaatc acaaccacat
     4201 cttagatagt tgaaatgact ttagataaca tcaattgaga tgagcttaat catgtcaaag
     4261 ctaaaagtgt caccatgaac gacaattctt aagcaaatca cgtgatatag atccacgaat
     4321 aaccaccatt tgatgctcga ggcaagtaat gtgtgtaaaa aaatgcgtta ccaccatcca
     4381 atgcagaccg atcttctacc cagaatcaca tatatttatg taccgagtac cttttttcta
     4441 tcttccaatt gcttctccca tatgattgtc tccgtaagct cgaaatttct aagttggatt
     4501 ttaatcttca cgcaggatga cagttcgatg agcttctgag gagtgtttag aacataatca
     4561 tttatccatg gtctatctct tcttgtcgct ttttctcctc gatagaacct aaataaaacg
     4621 agctctcgag aacccttaat ataacttcgt ataatgtatg ctatacgaag ttattaggtg
     4681 atatcagatc cactagtggc ctatgcggcc gcggatctgc cggtctccct atagtgagtc
     4741 gtattaattt cgataagcca ggttaacctg cattaatgaa tcggccaacg cgcggggaga
     4801 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4861 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4921 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4981 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5041 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5101 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5161 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5221 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5281 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5341 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5401 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5461 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5521 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5581 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5641 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5701 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5761 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5821 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5881 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5941 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6001 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6061 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6121 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6181 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6241 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6301 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6361 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6421 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6481 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6541 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6601 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6661 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6721 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     6781 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     6841 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     6901 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     6961 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat ggacatattg
     7021 tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat acgatttagg
     7081 tgacactata
//
