LOCUS       sequence-205181-        3391 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite gateway vector for 3' tagging with APEX2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3391)
  AUTHORS   Ariotti N, Rae J, Giles N, Martel N, Sierecki E, Gambin Y, Hall TE, 
            Parton RG
  TITLE     Ultrastructural localisation of protein interactions using 
            conditionally stable nanobodies.
  JOURNAL   PLoS Biol. 2018 Apr 5;16(4):e2005473. doi: 
            10.1371/journal.pbio.2005473. eCollection 2018 Apr.
  PUBMED    29621251
REFERENCE   2  (bases 1 to 3391)
  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: "PLoS 
            Biol."; date: "2018-04-5"; volume: "16(4):e2005473. doi"; pages: " 
            10.1371/journal.pbio.2005473. eCollection 2018 Apr"
COMMENT     Sequence Label: p3E-APEX2
FEATURES             Location/Qualifiers
     source          1..3391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(624..747)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             750..1496
                     /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"
     protein_bind    complement(1500..1595)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1632..1651)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1633..1651)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1656..1672)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1656..1672)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1774..1793)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1785..2594
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1852..1871)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2741..3329
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3230..3249
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagtggctg gaaagtctta cccaactgtg agtgctgatt
      781 accaggacgc cgttgagaag gcgaagaaga agctcagagg cttcatcgct gagaagagat
      841 gcgctcctct aatgctccgt ttggcattcc actctgctgg aacctttgac aagggcacga
      901 agaccggtgg acccttcgga accatcaagc accctgccga actggctcac agcgctaaca
      961 acggtcttga catcgctgtt aggcttttgg agccactcaa ggcggagttc cctattttga
     1021 gctacgccga tttctaccag ttggctggcg ttgttgccgt tgaggtcacg ggtggaccta
     1081 aggttccatt ccaccctgga agagaggaca agcctgagcc accaccagag ggtcgcttgc
     1141 ccgatcccac taagggttct gaccatttga gagatgtgtt tggcaaagct atggggctta
     1201 ctgaccaaga tatcgttgct ctatctgggg gtcacactat tggagctgca cacaaggagc
     1261 gttctggatt tgagggtccc tggacctcta atcctcttat tttcgacaac tcatacttca
     1321 cggagttgtt gagtggtgag aaggaaggtc tccttcagct accttctgac aaggctcttt
     1381 tgtctgaccc tgtattccgc cctctcgttg acaaatatgc agcggacgaa gatgccttct
     1441 ttgctgatta cgctgaggct caccaaaagc tttccgagct tgggtttgct gatgcctaac
     1501 aactttatta tacaaagttg gcattataaa aaagcattgc ttatcaattt gttgcaacga
     1561 acaggtcact atcagtcaaa ataaaatcat tatttggagc tccatggtag cgttaacgcg
     1621 gccgcgatat cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct
     1681 ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa
     1741 caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac
     1801 gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     1861 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg
     1921 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     1981 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     2041 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc
     2101 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     2161 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     2221 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     2281 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     2341 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     2401 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     2461 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     2521 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     2581 atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga
     2641 cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc
     2701 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2761 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2821 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2881 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2941 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3001 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3061 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3121 tacagcgtga gcattgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3181 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3241 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3301 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3361 tggccttttg ctggcctttt gctcacatgt t
//
