LOCUS       sequence-145719-        1076 bp    DNA     linear   UNA 14-APR-2025
DEFINITION  Expression of cytosolic APEX2 in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1076)
  AUTHORS   Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, 
            Ting AY
  TITLE     Directed evolution of APEX2 for electron microscopy and proximity 
            labeling.
  JOURNAL   Nat Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179.
  PUBMED    25419960
REFERENCE   2  (bases 1 to 1076)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179."
COMMENT     Sequence Label: pTRC-APEX2
FEATURES             Location/Qualifiers
     source          1..1076
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        join(50..55,56..72,73..79)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    87..103
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     92..114
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             130..147
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             148..894
                     /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"
     primer_bind     complement(961..978)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
ORIGIN
        1 ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgt tgacaattaa
       61 tcatccggct cgtataatgt gtggaattgt gagcggataa caatttcaca caggaaacag
      121 accatggcac accaccacca ccaccacgga aagtcttacc caactgtgag tgctgattac
      181 caggacgccg ttgagaaggc gaagaagaag ctcagaggct tcatcgctga gaagagatgc
      241 gctcctctaa tgctccgttt ggcattccac tctgctggaa cctttgacaa gggcacgaag
      301 accggtggac ccttcggaac catcaagcac cctgccgaac tggctcacag cgctaacaac
      361 ggtcttgaca tcgctgttag gcttttggag ccactcaagg cggagttccc tattttgagc
      421 tacgccgatt tctaccagtt ggctggcgtt gttgccgttg aggtcacggg tggacctaag
      481 gttccattcc accctggaag agaggacaag cctgagccac caccagaggg tcgcttgccc
      541 gatcccacta agggttctga ccatttgaga gatgtgtttg gcaaagctat ggggcttact
      601 gaccaagata tcgttgctct atctgggggt cacactattg gagctgcaca caaggagcgt
      661 tctggatttg agggtccctg gacctctaat cctcttattt tcgacaactc atacttcacg
      721 gagttgttga gtggtgagaa ggaaggtctc cttcagctac cttctgacaa ggctcttttg
      781 tctgaccctg tattccgccc tctcgttgac aaatatgcag cggacgaaga tgccttcttt
      841 gctgattacg ctgaggctca ccaaaagctt tccgagcttg ggtttgctga tgcctaagat
      901 ccgctagagt cgacctgcag gcaagcttgg ctgttttggc ggatgagaga agattttcag
      961 cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg
     1021 cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgta
//
