Part:BBa_K1445001

Endogenous Type II CRISPR-Cas9 phagemid

The M13ori-pCas9 composite part consists of the M13 origin of replication (M13ori) and the pCas9 part (BBa_K1218011) containing a tracrRNA sequence, the cas9 gene, and a minimal CRISPR array.

M13ori (BBa_K1445000) is a 500bp noncoding sequence that is recognized by the gene II of the M13 phage, which results in the uptake of the plasmid containing the ori into an assembled phage coat. This part does not contain any complete phage genes so phage production requires a Helper Phagemid such as M13K07. There will be some packaging of Helper Phagemid but their packaging signal weakened so parts containing the M13ori are preferentially packaged.

pCas9 contains the native trcrRNA and promoter region upstream of the Cas9 protein. The type II Cas9 protein is the native, active version from streptococcus pyogenes with the exceptrion of a SNP to erase an illegal EcoRI cleavage site. Downstream of the Cas9 protein is a minimal CRISPR array. This minimal array only includes two CRISPR repeats flanking a spacer region. It is this spacer region that determines which DNA sequence is targeted by the Cas9 endonuclease. Spacer sequence can be easily replaced with the protocol found here.

Usage and Biology

Boulder_M13ori-pCas9_FIGURE_1.png

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 2179
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 4458
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 5400
  Illegal BsaI.rc site found at 5377