Part:BBa_K243018

His-DigA-Split Linker-Fok_i

This part unites some of our parts and their features.

Usage and Biology

This composite part is one part of our universal endonuclease and it needs another composite part like BBa_K243024 to built to a functional heterodimer. The DigA tag leads the part to DNA which is hybridized with an Digoxigenin labeled oligonucleotide. The split linker makes a distance from 51bp between the DigA and the linked Fok_i protein domain. The His-Tag serve as 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 flurescein 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 efficiently than the use of a combination of DigA tagged oligos with a construct containing Fok_a. 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]