Part:BBa_K2340000

dCAS13a linked to GFP with NLS

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 1112
  Illegal BglII site found at 2048
  Illegal BglII site found at 2312
  Illegal BglII site found at 2816
  Illegal BglII site found at 3302
  Illegal BglII site found at 3410
  Illegal BglII site found at 3740
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 662
  Illegal SapI.rc site found at 3421
  

Usage and Biology

dead CAS13a (or dCas13) with a double HEPN nuclease 1 and 2 inactive mutation, linked to a green fluorescent protein (wtGFP) via a linker sequence (ggctcctccggc). On the N-terminal and the C-terminal of the construct there are nuclear localization sequences (NLS; DNA sequence: cccaagaaaaaacgcaaggtg. Amino acid sequence: PKKKRKV) that will reduce background noise when expressed and used in mammalian cell lines.

When supplied with a guide RNA (gRNA), this enzyme will process it into a CRISPR RNA (crRNA). The crRNA contains a sequence that is complementary to the RNA it is targetting.

Validation

The part was validated with two screening primers 200bp up- and downstream of insert and then seperated on a 1% agarose gel using electrophoresis.

Figure 1: Agarose gel of a screening PCR of BBa_K2340000 in pSB1C3. We used two primer approximately 200bp up- and downstream of our insert