Part:BBa_K2761009

Cas1-Cas2 with Flag and Ha tags

CRISPR-Cas is a prokaryotic immune system that protects bacteria from phages and plasmids. With this complex, foreign DNA in the form of spacers is incorporated into the bacteria’s genome, specifically at the CRISPR locus. This acquisition of new DNA generates a memory of each “infection”.

Several Cas proteins are involved in this system, but Cas1 and Cas2 are the only proteins needed for acquisition of new spacers. These proteins have to detect the PAM sequence in order to enable their function as endonucleases, in other words, their activity to work as scissors that cut DNA sequences.

Crispr-cas complex is conformed by two dimers of cas1 and one dimer of cas2, forming a hexamer.

This cassette of expression of Cas1 and Cas2 proteins regulated by a T7 promoter induced by IPTG. Contains tags to extract and purified CAS proteins. Cas1 contains a Flag tag and Cas2 contains a HA tag. Each protein contains its own RBS and the construct contains only one T7 terminator.

Figure 1: Expresion of the Cas 1 and Cas 2 proteins, that form an hexamer with endonuclease activity

SJTU-BioX-Shanghai 2019's Improvement

We made a mutation inactivating the HindIII restriction digestionsite. See our improvement BBa_K2996800
Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 192
  Illegal XhoI site found at 985
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 913
  
• 1000
  COMPATIBLE WITH RFC[1000]