Part:BBa_K243010

His-FluA-Split Linker-Fok_i

This part unites some of our basic parts. The combination of a Histidin-Tag, an Anticalin-Tag and the linked protein domain Fok_a makes it possible to purify and detect the function of the protein. The fluoresceinA tag allows the measurement by quenching and the coupling to a fluorescein linked oligo.

Usage and Biology

This composite part is one part of our universal endonuclease and it needs another composite part like BBa_K243017 to built to a functional heterodimer. The FluA tag leads the part to DNA which is hybridized with a fluorescein labeled oligonucleotide. The Split Linker creates a distance of 51bp between the FluA and the linked Fok_i protein domain. The HisTag serves as a purification tag for Ni-NTA column purification.

Purification

We purified this part of our universal endonuclease by NiNTA column purification and proved the expression of the protein with a following Western Blot.

Western Blot: His-FluA-SplitLi-Fok_i in pEx;
lanes: NEB prestained marker broad range,
pool of elution fractions 2-5, empty lane, 3 positive controls

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]