pRibo-Sec-APEX2
(Plasmid #111699)

• Purpose: Theophylline-inducible expression of periplasmic APEX2 from a mycobacterial shuttle plasmid (episomal)
• Depositing Lab: Jessica Seeliger
• Publication: Ganapathy et al ACS Infect Dis. 2018 Apr 30. doi: 10.1021/acsinfecdis.8b00044.

Backbone

• Vector backbone: pRibo BsaHind
• Total vector size (bp): 5226
• Modifications to backbone: Derivative of pMV261with promoter modified to include theophylline-inducible riboswitch (AddGene #36251) See reference: Seeliger JC, Topp S, Sogi KM, Previti ML, Gallivan JP, Bertozzi CR. A riboswitch-based inducible gene expression system for mycobacteria. PLoS ONE. 2012;7(1):e29266. PMCID: 3261144
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: APEX2
• Insert Size (bp): 891
• Promoter: hsp60-ribo
• Tag / Fusion Protein:
  • V5 (N terminal on insert)

Cloning Information

• Cloning method: Ligation Independent Cloning
• 5′ sequencing primer: CTAGAGGTGACCACAACG
• 3′ sequencing primer: GATGCCTGGCAGTCGATC

Resource Information

• Supplemental Documents:
  • pRibo-Sec-APEX2.gb
• A portion of this plasmid was derived from a plasmid made by: Alice Ting (pcDNA3-mito-APEX, Addgene plasmid #42607)
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This plasmid is for theophylline-inducible expression of APEX2 in the periplasm of mycobacteria. It is episomal and multi-copy. The APEX gene was subcloned from pcDNA3-mito-APEX and subsequently mutated by site-directed mutagenesis to generate the optimized variant APEX2.

See also references: Rhee HW, Zou P, Udeshi ND, Martell JD, Mootha VK, Carr SA, Ting AY. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science. 2013;339(6125):1328-31. PMCID: 3916822.

Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, Ting AY. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods. 2015;12(1):51-4. PMCID: 4296904