Plasmid #1 encodes all three ttRNAP subunits with a C-terminal CL7 tag. Bsu36I/SwaI, HindIII/SfiI, and AhdI/SpeI restriction sites flank subunits 1, 2, and 3, respectively. Additionally, Bsu36I/SpeI excises the full 10kb restriction fragment containing all three subunits.
Transcription is induced with IPTG and driven by the T7 RNA polymerase. 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 is at the N-terminus of the first and second ttRNAP subunits. Two more are on either side of the RpoZ component of the third subunit.
Plasmid #1 also contains an N-terminal maltose-binding protein (MBP) tag upstream of the PSC cleavage site before the first subunit (RpoA). Additionally, there are a thioredoxin (Trx) tag and His6 tag at the N- and C-terminus of the second subunit, respectively. There is also an N-utilization substance (NusA) tag located at the N-terminus of the third subunit.