pBAD_GFP11_T7LysH17A
(Plasmid #59591)

• Purpose: (Empty Backbone) Expression vector for solubility screening of proteins or protein fragments using a split-GFP assay.
• Depositing Lab: Richard Kingston
• Publication: Bulloch et al Anal Biochem. 2014 Jul 10. pii: S0003-2697(14)00280-2. doi: 10.1016/j.ab.2014.06.021.

Backbone

• Vector backbone: pBAD-MCS
• Backbone manufacturer: Arie Geerlof - EMBL
• Backbone size (bp): 4332
• Modifications to backbone: The XhoI site of pBAD-MCS was first removed by site directed mutagenesis. Between the NcoI and HindIII sites of the modified pBAD-MCS vector a 132 bp DNA cassette was inserted for expression of cloned protein fragments with an N-terminal 6xHis-tag and a C-terminal GFP11 tag. Protein fragments can be cloned into this site using sticky end (SpeI and XhoI) or blunt end (PvuII) ligation. A 636 bp fragment of the plasmid pLysS, encompassing the T7 lysozyme gene, was ligated into a SphI site in a non-essential region of the vector. Finally the mutation H17A was introduced to the T7 lysozyme gene to reduce its amidase activity.
• Vector type: Bacterial Expression
• Promoter: pARA
• Tags / Fusion Proteins:
  • 6xHis-tag (N terminal on backbone)
  • GFP11 (C terminal on backbone)

Growth in Bacteria

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

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: ATG CCA TAG CAT TTT TAT CC
• 3′ sequencing primer: GAT TTA ATC TGT ATC AGG

Terms and Licenses

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