Part:BBa_K243013

His-FluA-Short Linker-Fok_i

This combination uses the benefits of an His-tag for purification. It is also linked with a FluroescineA-tag. The short linker (GlySerGlyGly)connects the parts FluA and Fok_i and brigs them in spatial proximity to each other and therefor in proximity to the DNA.

Usage and Biology

This composite part is one part of our universal endonuclease and it needs another composite part like BBa_K243017 to build a functional heterodimer. The FluA tag guides the part to DNA which is hybridized with a Fluorescein labeled oligonucleotide. The short linker creates a distance of 12bp between the FluA and the linked Fok_i protein domain. The His tag serves as a purification tag for a Ni-NTA column purification.

We applied the His tag to enable a simultaneous purification of constructs with a Strep tag. The used FluoresceinA tag allows the measurement by quenching and the coupling to a fluorescein linked oligo. Emanating from our 3D modeling this combination of FluA tagged oligo and the construct containing the protein domain Fok_i is more efficient than the use of a combination of DigA tagged oligo with a construct containing Fok_i. To get FluA and Fok_i together and therefore bring them in proximity to the DNA, we used the short linker. The linker itself has no influence on the connected parts.

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]