This plasmid contains the ttRNAP protein – a model DNA/RNA-binding multisubunit protein – with a C-terminal CL7 tag. All three ttRNAP subunits are encoded within the plasmid: subunits 1, 2, and 3 were inserted at Bsu36I/SwaI, HindIII/SfiI, and AhdI/SpeI restriction sites, respectively.
Transcription is driven by the T7 RNA polymerase, and the plasmid is designed for expression in E. Coli.
The CL7 tag for purification is fused to the C-terminus of the third subunit of the ttRNAP protein.
Plasmid 1 includes several PreScission protease (PSC) cleavage sites: one at the N-terminus of the first and second ttRNAP subunits, and two on either side of the RpoZ component of the third subunit.
In addition to the C-terminus CL7 tag, this plasmid also contains an N-terminal maltose-binding protein (MBP) tag upstream of the PSC cleavage site before the first subunit (RpoA); a thioredoxin (Trx) tag and His6 tag at the N- and C-terminus of the second subunit, respectively; and an N-utilization substance (NusA) tag at the N-terminus of the third subunit.