Part:BBa_K243024:Design
Strep-FluA-Middle Linker-Fok_a
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 278
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1075
Design Notes
The linker length and the choice of the purification tag in combination with the anticalin tag allowed us several combination possiblities, which we had to prove to achieve the best combiantion. We applied the Streptavidine tag to enable a simultaneous purification of constructs with a His tag. Strep tag also shows a higher affinity towards Strep-Tactin than His tag. In this case the purification with Strep tag is more specific. The used FluoresceinA tag allows the measurement by quenching and the coupling to a flurescein linked oligo. Emanating from our 3D modeling this combination of FluA tagged oligo and the construct containing the protein domain Fok_a is not as efficient as the use of a combination of DigA tagged oligo with a construct containing Fok_a. To avoid interactions between the FluA tag with the connected protein domain Fok_a we applied the Middle Linker. The Linker itself has no influence on the connected parts. We decided to use the Middle Linker for this construct to prove the optimal distance between the parts. It is important that the used linker has a certain flexibility and is long enough to avoid steric interferences between the parts. If the linker is too long it might cause the instability of the whole construct
Source
Combined by serial clonig steps.