Part:BBa_K5247135:Design
DNA fragment for CFTR-specific pegRNA screening
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 933
Illegal PstI site found at 980 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 933
Illegal PstI site found at 980 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 933
Illegal PstI site found at 980 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 933
Illegal PstI site found at 980 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
In addition to the introduction of the three-base-pair deletion, the intron sequence was further altered with the objective of enhancing the comparability of the system to the CFTR genomic context. Specifically, 27 base pairs were replaced downstream of the splice site with a sequence derived from the CFTR gene in the region of the F508del mutation. This modification guarantees that the gRNA spacer employed in our system is identical to the one found in the actual genomic context of the CFTR mutation. The only notable differences between the system and the genomic sequence are observed in the RTT (Reverse Transcript Template) and PBS (Primer Binding Site), which have been calibrated with silent mutations to maintain comparability in GC content with the native CFTR gene. These silent mutations do not affect the encoded protein but optimise the system's mimicry of the CFTR gene.
Source
Synthetic