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%.

Lure sequence upstream of eGFP.png

Fig:Schematic diagram of this part

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 66
    Illegal NheI site found at 89
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 834
    Illegal XhoI site found at 843
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None