pET21a Mo-iPGM-bio
(Plasmid #179614)

• Purpose: Expresses M. orale iPGM containing a C-terminal biotinylation site and His tag in E. coli
• Depositing Lab: James Inglese
• Publication: van Neer et al ACS Chem Biol. 2022 Aug 19;17(8):2284-2295. doi: 10.1021/acschembio.2c00403. Epub 2022 Jul 29.

Backbone

• Vector backbone: pET21a
• Backbone manufacturer: EMD Biosciences
• Backbone size w/o insert (bp): 5443
• Total vector size (bp): 6990
• Modifications to backbone: 1. Bm-iPGM-AviTag construct in pET21a was modified using site directed mutagenesis to remove the Xho restriction site located after AviTag and generate a universal backbone containing Avitag for easy cloning in frame with His tag. The resulting construct is as follows: BamH1 – Bm-iPGM– Xho1-AviTag-no Xho1-6xHis-stop 2. Bm-iPGM-AviTag is removed using restriction enzymes BamH1/Xho1 and Mo-iPGM (synthetized by BioBasic) is cloned into BamH1/Xho1 restriction sites. The resulting construct is as follows: BamH1 – Mo-iPGM– Xho1-AviTag-no Xho1-6xHis-stop
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: M. orale iPGM bio-6His
• Alt name: Mo iPGM bio
• Alt name: 2,3-bisphosphoglycerate-independent phosphoglycerate mutase
• Species: Mycoplasma orale
• Insert Size (bp): 1547
• GenBank ID: NZ_LR214940.1
• Promoter: T7
• Tags / Fusion Proteins:
  • biotinylation sequence (C terminal on insert)
  • His tag (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BamH1 (not destroyed)
• 3′ cloning site: Xho1 (not destroyed)
• 5′ sequencing primer: T7 promotor primer #69348-3
• 3′ sequencing primer: T7 terminator primer #69337-3

Terms and Licenses

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