Device
Part:BBa_K4170016:Design
Designed by: Alexandros Giannopoulos Dimitriou Group: iGEM22_Thessaloniki_Meta (2022-09-27)
SUMO-LbuCas13a coding device under T7 promoter
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1453
Illegal BglII site found at 5047 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1421
Illegal AgeI site found at 1910
Illegal AgeI site found at 3125 - 1000COMPATIBLE WITH RFC[1000]
Cloning strategy
For the final assembly of the PCR amplified genetic elements into pSB1C3 plasmid we followed the Golden Gate-based ‘SevaBrick Assembly’ method and the SEVA 3.1 platform. As for all our final composite parts, the initial steps of our cloning strategy constitute the PCR amplification of different genetic elements, followed by the efficient assembly of the PCR amplified genetic products into pSB1C3 backbone. The cloning process is described in detail below:
Step 1
- PCR amplification with T7 P0 FWD and T7 P0 RVS primers using the pGJK_His-SUMO-LbuCas13a as a template. This PCR produces the P0 part ready for Golden Gate assembly.
- PCR amplification with T7 P1 FWD and T7 P1 RVS primers using the pGJK_His-SUMO-LbuCas13a as a template. This PCR produces the P1 part ready for Golden Gate assembly.
- PCR amplification with P2 FWD and P4 RVS primers using the cloned cas13a repos plasmid as a template. This PCR produces the P2-P4 part 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 P0, P1, P2-P4 parts with the linearized pSB1C3 vector for the efficient construction of the SUMO-LbuCas13a coding sequence under the transcriptional control of the T7 promoter.