Coding

Part:BBa_K4170043:Design

Designed by: Alexandros Giannopoulos Dimitriou and Katerina Saiti   Group: iGEM22_Thessaloniki_Meta   (2022-09-27)


crRNA targeting the miR-17-5P (mismatch 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.
  • PCR amplification with 5pm spacer FWD interchangeable and spacer RVS standard primers using the PSB1C3 plasmid as a template. This PCR produces the 5pm-spacer part ready for Golden Gate assembly.

Step 2 Golden Gate assembly of the PCR amplified loop part and 5pm-spacer part for the efficient construction of the crRNA-5pm coding sequence under the transcriptional control of the T7 promoter.


Source

pp

References