Device

Part:BBa_K4170017:Design

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


LbuCas13a coding device under T7 promoter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1453
    Illegal BglII site found at 4750
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1421
    Illegal AgeI site found at 2828
  • 1000
    COMPATIBLE WITH RFC[1000]


Cloning strategy

Step 1

  • PCR amplification with SUMOLESS FWD and Cas13a P4 RVS primers using the final T7-SUMO-LbuCas13a as a template. This PCR produces the SUMOLESS-Cas13a part ready for Golden Gate assembly.
  • PCR amplification with T7 P0 FWD and SUMOLESS RVS primers using the final T7-SUMO-LbuCas13a as a template. This PCR produces the SUMOLESS-T7 ready for Golden Gate assembly.
  • PCR amplification with Ev and Pv standard primers from Basic SevaBrick Assembly [seva 3.1] using the Bba_J364007 part of the 2022 DNA distribution Kit. This PCR produced the pSB1C3 backbone linearized and ready for Golden Gate assembly.

Step 2

Golden Gate assembly of the PCR amplified SUMOLESS-Cas13a and SUMOLESS-T7 parts with the linearized pSB1C3 vector for the efficient construction of the SUMOLESS-LbuCas13a coding sequence under the transcriptional control of the T7 promoter.


Source

This plasmid has been constructed from pGJK_His-SUMO-LbuCas13a plasmid, deposited by Jennifer Doudna and her colleagues at Addgene plasmid repository

References