Part:BBa_K243010:Design

His-FluA-Split Linker-Fok_i

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]

Design Notes

The cloning steps were planned theoretically before we started the work in the wet lab. The combination of HisTag linked with a FluA tag connected by the Split Linker approved as a good way to express the inactive protein domain of our universal endonuclease.

part of universal restriction enzyme. Blue: DNA strand; Red: 16bp long Oligos, tag as indicated in the picture.
Ochre: Fluorescein A binding lipocalin;light Blue: inactive FokI cleavage domain;

We used the split linker because it is an improved part of the team [http://2008.igem.org/Team:Freiburg Freiburg08] and a suitable linker for fusion proteins. The properties of the split linker are a good compromise between the stability and the distance for the connection between protein domain Fok_a and the anticaline tag.The dimerization partner to this part needs another combination of anticaline and the protein domain Fok_a, because the labeled oligonucleotides had different anticalins. The parts are fused according to RFC 25.
Commented GenBank file

Source

Combined the parts by serial cloning steps.