Part:BBa_K3171177
HisD 3000bp Homologous part 1
The variable components needed for a CRISPR mediated knockout are gRNA and a sequence for homologous recombination. gRNA and the homologous part have to be engineered strictly depending on the target. In this case, all parts were designed with the aim to knockout HisD in V. natriegens.
We designed 3 homologous parts Part:BBa_K3171177, Part:BBa_K3171178, Part:BBa_K3171179 that serve as a template for homologous recombination once the Cas9 enzyme has cut the target site. Consequently, it is designed according to the up- and downstream regions of the targeted sequence. This part has a total length of 3 kbp. 1.5 kbp Part:BBa_K3171178 for each up- and downstream of HisD and 600 bp Part:BBa_K3171179 length were achieved using PCR and respective primers.
The gRNA enables the Cas9 enzyme to recognize and cut at the correct site. The Cas9 enzyme forms a complex with the gRNA and can then find the target sequence based on complementary base pairing. Parts Part:BBa_K3171180, Part:BBa_K3171181, Part:BBa_K3171182 have been designed to select suitable target site, considering e.g. possible off-targets or location within coding region. These parts have been ordered as single stranded oligonucleotides, annealed and ligated into a vector encoding the necessary compounds of CRISPR machinery.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 689
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1472
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 689
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 689
Illegal AgeI site found at 1885
Illegal AgeI site found at 2788 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 807
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