Part:BBa_K1160000

coding sequence of Cas9 from CRISPR system type II

CRISPR (clustered regularly interspaced short palindromic repeat) is an adaptive immune system provide protection against mobile genetic element by guided specific DNA cleavage. Cas9 (CRISPR-associated protein 9) belongs to type II CRISPR system and contains HNH motif and three RuvC-like motifs, homologous to HNH and RuvC endonuclease, respectively. Cas9 has strong DNA cleavage activity, thus it showed potential use in gene editing.

This part contains the coding sequence of cas9 from Streptococcus pyogenes (UniProtKB AC number:Q99ZW2). Some "redundant" sequence was added to ensure the Cas9's normal function or just for the convenience to detect it in cell.

The cas9 protein must be guided by two RNAs, crRNA(CRISPR RNA) and tracrRNA(trans-activating crRNA), to complete its specific DNA cleavage function. We build another part-a chimeic RNA-that combines the feature two RNAs. For more details, refer to part BBa_K1160001.

With this part, you can 1)directly use it to express cas9 protein in cell or 2)use T7 RNA polymerase to get cas9's mRNA, which can be further modified to prepare authentic mRNA that can be used for translation in vivo.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 249
  Illegal PstI site found at 3115
  Illegal PstI site found at 4561
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 895
  Illegal SpeI site found at 249
  Illegal PstI site found at 3115
  Illegal PstI site found at 4561
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 12
  Illegal BglII site found at 1154
  Illegal BglII site found at 4479
  Illegal BamHI site found at 2243
  Illegal XhoI site found at 2041
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 249
  Illegal PstI site found at 3115
  Illegal PstI site found at 4561
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 249
  Illegal PstI site found at 3115
  Illegal PstI site found at 4561
  Illegal NgoMIV site found at 3643
  Illegal NgoMIV site found at 5515
  Illegal NgoMIV site found at 6775
  Illegal AgeI site found at 5068
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 5050
  Illegal BsaI.rc site found at 882
  Illegal BsaI.rc site found at 8303
  Illegal SapI site found at 2556
  Illegal SapI site found at 7220
  Illegal SapI.rc site found at 2062
  Illegal SapI.rc site found at 2800
  Illegal SapI.rc site found at 3777