plasmid 21 option 1
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Plasmid #21

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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.

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 N-terminal CL7 tag is upstream of several protease cleavage sites and a His8 tag.
Cleavage Site(s)

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.
Other Tags

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.
Cloning Options


    1. HindIII/XhoI Insertion Site – Trx | CL7 | PSC | 44-Amino Acid Linker | PSC | His8 Tag | SUMO | 13-Amino Acid Linker | SRT | Gene of Interest

    1. KpnI/XhoI Insertion Site – Trx | CL7 | PSC | 44-Amino Acid Linker | PSC | His8 Tag | SUMO | 3-Amino Acid Linker | Gene of Interest

    1. 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: