Composite
Part:BBa_K3365062
Designed by: Zou Aixuan Group: iGEM20_SJTU-BioX-Shanghai (2020-10-23)
First lure sequence upstream of eGFP
This part is an off-target detecting platform for dCas9-ω. We put this part in the plasmid and transform it into △rpoZ-MG1655, a strain of E.coli that is koncked out rpoZ. If dCas9-ω is binding to the lure sequence, the subnit Omega fused to dCas9 C-terminal can activate the expression of reporter by recruiting the RNAP holoenzyme and the cell would emit green fluorescence,which means dCas9-ωis off-target, else the weak promoter and the lack of rpoZ makes the reporter cannot express or just have a quite low expression level.
According to the refrence, the off-target rate of the lure sequence in this part is 0.08286%.
Fig:Schematic diagram of this part
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 66
Illegal NheI site found at 89 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 834
Illegal XhoI site found at 843 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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Categories
Parameters
None |