Part:BBa_K243018

His-DigA-Split Linker-Fok_i

This combination uses the benefits of a His tag (Polyhistidin-tag) for purification. It is also linked with a DigoxigeninA tag DigA. The Split Linker connects the parts and adds additional space between them to guarantee the independent function of DigA tag and the protein domain Fok_i.

Usage and Biology

This composite part is one part of our universal endonuclease and it needs another composite part like BBa_K243024 to build a functional heterodimer. The DigA tag guides the part to DNA which is hybridized with a Digoxigenin labeled oligonucleotide. The Split Linker creates a distance of 51bp between the DigA and the linked Fok_i protein domain. The His Tag serves as a purification tag for Ni-NTA column purification.

We applied the His tag to enable a simultaneous purification of constructs with a Strep tag. The used DigoxigeninA tag allows the coupling to an fluorescein linked oligo. Emanating from our 3D modeling this combination of DigA tagged oligos and the construct containing the protein domain Fok_i is not that efficient as the use of a combination of FluA tagged oligos with a construct containing Fok_i. To avoid interactions between the DigA tag with the connected protein domain Fok_i we applied the Split Linker. The linker itself has no influence of the connected parts. We decided to use the Split Linker for this construct to get a longer distance between DigA tag and Fok_i to guarantee the independent function of both parts. This linker is an improved part of the team Freiburg08 and it is a suitable linker for fusion proteins.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 272
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]