pET30b-5M SrtA with TEV site
(Plasmid #86962)

• Purpose: Expresses calcium-dependent Sortase A with a TEV cleavage site proximal to the C-terminal histidine tag
• Depositing Lab: Ron Bose
• Publication: Sarpong et al Anal Biochem. 2017 Mar 15;521:55-58. doi: 10.1016/j.ab.2017.01.008. Epub 2017 Jan 11.

Backbone

• Vector backbone: pET30b
• Backbone size w/o insert (bp): 5400
• Total vector size (bp): 5892
• Modifications to backbone: C-terminal 6x histidine tag; C-terminal TEV site
• 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: S.aureus SrtA 5M
• Alt name: Sortase A pentamutant
• Species: Staphylococcus aureus
• Insert Size (bp): 492
• Mutation: P94R, D160N, D165A, K190E, K196T
• Promoter: T7
• Tags / Fusion Proteins:
  • TEV site (C terminal on backbone)
  • 6x Histidine tag (C terminal on backbone)

Cloning Information

• Cloning method: Ligation Independent Cloning
• 5′ sequencing primer: T7
• 3′ sequencing primer: T7-terminator

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

Pentamutant sortase A was originally described by David R. Liu's laboratory (Harvard University) in: A General Strategy for the Evolution of Bond-Forming Enzymes Using Yeast Display. Chen I, Dorr BM, Liu DR. Proc Natl Acad Sci U S A. 2011 Jul 12;108(28):11399-404 (PubMed ID: 21697512). As noted in this publication, the cloned sortase A gene lacks the N-terminal 59 amino acids, which do not impact catalytic activity, but these amino acids are still included in the numbering for the mutations.