Plasmid #3

Plasmid #3 serves as a template for tagging target proteins with N-terminal CL7 tag. It has three options for the 5’ placement of target genes (see cloning options below), and XhoI is the 3’ cloning site.  All three options require a stop codon encoded in the 3’ cloning primer.  There is an existing 1711 base pair restriction fragment inserted at the HindIII/XhoI restriction sites. 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 N-terminus of the target protein, upstream of the SUMO cleavage site. CLEAVAGE SITE(S): Plasmid #3 includes two protease sites: a SUMO and Sortase A (SRT) cleavage site exist between the CL7 binding site and downstream target protein. OTHER TAGS: The plasmid includes an N-terminal thioredoxin (Trx) tag upstream of the CL7 tag. CLONING OPTIONS: 1. HindIII/XhoI Insertion Site – Trx | CL7 | SUMO | SRT | Gene of Interest 2. KpnI/XhoI Insertion Site – Trx | CL7 | SUMO | Gene of Interest 3. Bsu36I/XhoI Insertion Site – Trx | CL7 | SUMO | Gene of Interest EXPIRATION: 6 months from receipt when stored as directed. Plasmid guide with plasmid sequence file is available at Trialtusbioscience.com.