Plasmid #1

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. EXPRESSION: Transcription is induced with IPTG and driven by the T7 RNA polymerase. The plasmid is designed for expression in E. coli. PEPTIDE TAG: The CL7 tag for purification is fused to the C-terminus of the third subunit of the ttRNAP protein. CLEAVAGE SITE(S): 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. OTHER TAGS: 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. EXPIRATION: 6 months from receipt when stored as directed. Plasmid guide with plasmid sequence file is available at Trialtusbioscience.com