sortase A pentamutant (eSrtA) in pET29
(Plasmid #75144)

• Purpose: evolved sortase A (eSrtA) pentamutant with improved kinetics and activity
• Depositing Lab: David Liu
• Publication: Chen et al Proc Natl Acad Sci U S A. 2011 Jul 12;108(28):11399-404. doi: 10.1073/pnas.1101046108. Epub 2011 Jun 22.

Backbone

• Vector backbone: pET29
• Backbone manufacturer: Novagen
• Backbone size w/o insert (bp): 5370
• 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: sortase A pentamutant
• Alt name: 5M SrtA
• Alt name: evolved sortase A (eSrtA)
• Species: Staphylococcus aureus
• Insert Size (bp): 441
• Mutation: P94R, D160N, D165A, K190E, K196T. Deleted amino acids 1-59 (which do not affect catalytic activity).
• Promoter: T7
• Tag / Fusion Protein:
  • 6x His (C terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NdeI (not destroyed)
• 3′ cloning site: XhoI (not destroyed)
• 5′ sequencing primer: T7
• 3′ sequencing primer: T7-term

Resource Information

• Supplemental Documents:
  • Annotated GenBank file
• Addgene Notes:
  • Addgene Diagnostic Digest
• Articles Citing this Plasmid:
  • 25 References

Terms and Licenses

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

Depositor Comments

This plasmid has been found to be somewhat unstable and prone to concatenation. Concatenation often does not impact plasmid function, but may reduce transformation efficiencies. You may need to screen multiple colonies to isolate the monomeric version of this plasmid. If you still have trouble isolating the monomeric version, you might consider linearizing, gel extracting, re-ligating, and transforming the plasmid.