Part:BBa_K4887005

Cas9

It is the gene of Cas9 (CRISPR-associated nuclease 9), a dual RNA-guided DNA endonuclease enzyme associated with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) adaptive immune system in Streptococcus pyogenes.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 907
  Illegal PstI site found at 2329
  Illegal PstI site found at 2533
  Illegal PstI site found at 2563
  Illegal PstI site found at 3775
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 907
  Illegal PstI site found at 2329
  Illegal PstI site found at 2533
  Illegal PstI site found at 2563
  Illegal PstI site found at 3775
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 368
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 907
  Illegal PstI site found at 2329
  Illegal PstI site found at 2533
  Illegal PstI site found at 2563
  Illegal PstI site found at 3775
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 907
  Illegal PstI site found at 2329
  Illegal PstI site found at 2533
  Illegal PstI site found at 2563
  Illegal PstI site found at 3775
  Illegal NgoMIV site found at 1195
  Illegal NgoMIV site found at 2299
  Illegal NgoMIV site found at 2372
  Illegal NgoMIV site found at 2857
  Illegal NgoMIV site found at 3766
  Illegal NgoMIV site found at 4227
  Illegal NgoMIV site found at 4246
  
• 1000
  COMPATIBLE WITH RFC[1000]

5.5 Results:

(1) PCR detection for gene Cas9.

Genome DNA of the transgenic lines was extracted from leaves of these two regenerated seedlings. PCR detection on the genomes was performed by using primers designed based on the Cas9 protein gene. As the result, the transgenic lines 23216004 and 23216005 were further validated (Fig. 1).
The sequences of the primers were as bellow:

• CAS9-F: 5’-atggactataaggaccacgacgg-3’
• CAS9-R: 5’-ttgtcgcctcccagctgagacag-3’

Fig. 1 Detection results of PCR for gene Cas9.