Plasmid #21 enables the tagging of target proteins with N-terminal CL7 and His8. There are three options for the 5’ placement of target genes (see cloning options below). XhoI is the 3’ cloning site. All three options require a stop codon encoded in the 3’ cloning primer.
Transcription is induced with IPTG and driven by the T7 RNA polymerase. The plasmid is designed for expression in E. coli.
The N-terminal CL7 tag is upstream of several protease cleavage sites and a His8 tag.
Plasmid #21 contains two PreScission protease (PSC) cleavage sites between the CL7 and His8 tags. Downstream of the His8 tag are SUMO and Sortase A cleavage sites.
The coding region begins with an N-terminal Trx tag and includes a His8 tag between the PSC and SUMO cleavage sites, upstream of the target protein.
- HindIII/XhoI Insertion Site – Trx | CL7 | PSC | 44-Amino Acid Linker | PSC | His8 Tag | SUMO | 13-Amino Acid Linker | SRT | Gene of Interest
- KpnI/XhoI Insertion Site – Trx | CL7 | PSC | 44-Amino Acid Linker | PSC | His8 Tag | SUMO | 3-Amino Acid Linker | Gene of Interest
- Bsu36I/XhoI Insertion Site – Trx | CL7 | PSC | 44-Amino Acid Linker | PSC | His8 Tag | SUMO | Gene of Interest
The Bsu361/XhoI insertion scheme maintains the Gene of Interest’s wildtype sequence without adding any extra residues. The N-terminus of the Gene of Interest must include the following sequence to complete the SUMO C-terminal sequence: