Part:BBa_K4170019:Design

crRNA targeting the miR-17-3P (standard design) under T7 promoter

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 71
  

Cloning strategy

For the final construction of the plasmid, PCR amplified genetic elements were assembled following the Golden Gate-based ‘SevaBrick Assembly’ method. The cloning process is described in detail below:

Step 1

• PCR amplification with loop RVS standard and loop FWD standard primers using the PSB1C3 plasmid as a template. These primers produce the loop sequence of the crRNA incorporated with the CmR sequence (confers resistance to chloramphenicol) of the PSB1C3 plasmid. This PCR produces the loop part ready for Golden Gate assembly. The loop part is ready to use for any crRNA constructed with this method as the stem-loop sequence is universal for all crRNAs.
• PCR amplification with 3ps spacer FWD interchangeable and spacer RVS standard primers using the PSB1C3 plasmid as a template. This PCR produces the 3ps-spacer part ready for Golden Gate assembly.

Step 2

Golden Gate assembly of the PCR amplified loop part and 3ps-spacer part for the efficient construction of the crRNA-3ps coding sequence under the transcriptional control of the T7 promoter.

Source

pp