pMC_0_3-5_1_crRNA-GFP_dropout
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1186
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1186
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1186
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 242
Design Notes
The crRNA part had to be ordered in 4 different parts, because there are direct repeats in between the different gRNA spaceholders. These 4 parts were ordered with BsaI overhangs that were cut out with a Golden Gate reaction and the final construct was assembled via annealing of the resulting overhangs. We use a GFP cassette as a placeholder for the gRNA, that can be cut out via BsmbI digest. The part reaches from the beginning of the RBS overhang to the end of the Terminator.
Source
The crRNA was synthesized in 4 parts via IDT gene synthesis.