Part:BBa_K578002

T7LRSR (Listeria innocua CLIP11262)

This brick, T7LRSR, is the starting point for a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) array based around Listeria Innocua CLIP11262. The sequence of the part is composed of a T7 promoter, Lac Operator, Leader Sequence (from Listeria Innocua CLIP11262), Palindromic Repeat, Spacer (Targeted to sequence with correct protospacer and 'NGG; Protospacer-adjacent motif), followed by a palindromic repeat. After the aforementioned sequences there is a pseudospacer with the standard BioBrick suffix (with SpeI and PstI Sites). The suffix allows for parts (eg.Repeat.Spacer Repeat, RSR, formated BioBricks) to be added onto the distal end of the CRISPR array.

Note: This Part also requires two parts not submitted to the registry, tracrRNA, which forms a non-coding RNA that initiates formation of mature CRISPR RNA by engaging RNaseIII and Cas9, and the coding region for Cas9 itself.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Confirmation of Part Length

PCR of BBa_K578002 using Primers VF2 (BBa_G00100) and VFR (BBa_G00101) from pSB1C3. The part is 291bp plus 42bp for the Standard BioBrick prefix and suffix. Note: The primers add approximatellly 200bp to the BioBrick's size.

Use of this BioBrick

This part has been [http://2011.igem.org/Team:Arizona_State/Project/L_innocua#Construction successfully ligated] into pRSF Duet along with Cas9 and tracrRNA precursor, ready for inducible CRISPR-Cas expression.