pET28a-hfYFP-TEV-Bcx
(Plasmid #191195)

• Purpose: Fusion of hfYFP and Bacillus circulans xylanase separated by a TEV cleavage site for fluorescence-assisted protein purification
• Depositing Lab: Gregory Petsko
• Publication: Campbell et al Nat Methods. 2022 Nov 7. pii: 10.1038/s41592-022-01660-7. doi: 10.1038/s41592-022-01660-7.

Backbone

• Vector backbone: pET28a
• Total vector size (bp): 6631
• Modifications to backbone: Thrombin cleavage site eliminated.
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For protein production, transform the plasmid into BL21(DE3) cells and induce expression using IPTG.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: hfYFP & Bcx fusion
• Alt name: Hyperfolder YFP
• Alt name: Bacillus circulans xylanase
• Species: Synthetic
• Insert Size (bp): 1395
• Promoter: T7 promoter
• Tags / Fusion Proteins:
  • Bacillus circulans xylanase (C terminal on insert)
  • 6xHis (N terminal on insert)

Cloning Information

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

Terms and Licenses

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

Depositor Comments

Please refer to the manuscript to understand how to use this expression/purification system.

Bacillus circulans xylanase (synonymous with Niallia circulans endo-1,4-beta-xylanase) was synthesized from UniProtKB/Swiss-Prot: P09850.1 amino acid sequence using a bacterial codon set.