VopL/F constructs were prepared by infusion (Takara Bio Inc.) after PCR amplification (iProof; Bio-Rad Laboratories) from plasmid DNA for VopL and from codon-optimized plasmid DNA for VopF (provided by E. Kerkhoff, University of Regensburg, Regensburg, Germany), with a 6xHis tag included in the reverse primer. PCR products were cloned into the SNAP-tag-T7-2vector (New England Biolabs, Inc.) at the XmaI/PacI sites, but the PacI site is not maintained. A flexible linker (GGSGGS) was included in the forward primer sequences of the SNAP constructs between the SNAP and the VopL/F DNA sequences.
Snap tag t7 2vector
Manufactured by New England Biolabs
SNAP-tag-T7-2vector is a plasmid vector that expresses a SNAP-tag fusion protein under the control of a T7 promoter. The SNAP-tag is a self-labeling protein tag derived from the human DNA repair protein O6-alkylguanine-DNA alkyltransferase. This vector can be used for the expression and purification of SNAP-tag fusion proteins in E. coli.
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Lab products found in correlation
2 protocols using snap tag t7 2vector
1
Production and Cloning of VopL/F Constructs
2
Cloning Expression Plasmids for Actin-Binding Proteins
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Expression plasmids for FH1-FH2, FH2 (Li et al., 2010) , mDia1 FH1-FH2-C (Moseley et al., 2006) , ABP29 (Xiang et al., 2007) , AtPRF2 (Wang et al., 2009) , AtPRF4 (Ye et al., 2009) , LlPRF1 (Vidali and Hepler, 1997) , and human profilin (Kovar et al., 2006) have been described previously. The SNAP coding region was PCR amplified from the SNAP-tag-T7-2-vector (NEB, Ipswitch, MA), and combined with FH1-FH2, FH2, ABP29, and mDia1-FH1-FH2-C by fusion PCR with a GGSGGS flexible linker between them. All these PCR products were cloned into the Nde1/Not1 sites of pET30a expression vectors (Novagen, San Diego, CA, USA).
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