MZB211
(Plasmid #228494)

• Purpose: 10xHis-SUMO (Smt3) tagged Ssod7-fused Pfu Polymerase with fidelity enhancing mutation (E. coli based protein expression)
• Depositing Lab: Richard Baker
• Publication: Molecular Toolbox (unpublished)

Backbone

• Vector backbone: pET29b
• Backbone size w/o insert (bp): 5630
• Total vector size (bp): 8144
• Vector type: Bacterial Expression

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: pol
• Alt name: Pfu
• Species: Synthetic; Pyrococcus furiosus
• Insert Size (bp): 2514
• Mutation: A408S
• GenBank ID: BAA02362.1
• Promoter: T7 Promoter
• Tag / Fusion Protein:
  • 10xHis-SUMO(Smt3) (N terminal on backbone)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: T7 promoter
• 3′ sequencing primer: T7 terminator

Resource Information

• Supplemental Documents:
  • MZB211.gb
• A portion of this plasmid was derived from a plasmid made by: Brian Strahl

Terms and Licenses

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

Depositor Comments

Bacterial expression of 10xHis-SUMO tagged Pfu polymerase fused to Sso7d processivity domain (commercially sold as Phusion Polymerase). Polymerase domain contains the A408S amino acid substitution for enhanced fidelity.

Kennedy EM, Hergott C, Dewhurst S, Kim B. The mechanistic architecture of thermostable Pyrococcus furiosus family B DNA polymerase motif A and its interaction with the dNTP substrate. Biochemistry. 2009 Dec 1;48(47):11161-8. doi: 10.1021/bi9010122. PMID: 19817489; PMCID: PMC3097049.