Part:BBa_K2201032

sgRNAs MAX target + mRFP-sacB fusion protein

sgRNAs MAX target + mRFP-sacB fusion protein

This composite part is the second plasmid used for the two-plasmid retention system to preserve the unnatural base pair (UBP). It consists of the Biobricks BBa_K2201077 (five sgRNAs) and BBa_K2201017 (mRFP-sacB fusion protein). An Oligo containing the UBP can be assembled with this composite part BBa_K2201032 via Gibson Assembly. Therefore this part needs to be linearized via PCR. The resulting overhangs would need to overlap with the target oligo.
We used primers to linearize the BBa_K2201032 backbone so that an successful Gibson Assembly with the UBP-oligo leads to a frame shift of the sacB gene. Hence, the organism is now able to grow in the presence of sucrose.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1327
  Illegal NheI site found at 1350
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1936
  Illegal AgeI site found at 2048
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 923