Part:BBa_K4654018:Design

T7-Promoter_Spacer1-Li-+II_Riboswitch-Spacer2-5'nhaA_nanoLuc

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 30
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 30
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 30
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 30
  Illegal NgoMIV site found at 123
  Illegal NgoMIV site found at 278
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

During the design process we made sure to include the spacer sequences up- and downstream of the riboswitch because they influence the folding of the riboswitch. We originally did not know where the RBS was located in Spacer Region 2 so we had to verify the location by asking the authors.

Source

The T7 promoter is derived from the T7 phage. The riboswitch can be found in certain bacteria, the sequence we are using was created by White et. al. by creating a consenus sequence from different sequences of the riboswtich family¹. nanoLuc originates from the deep sea shrimp Oplophagus gracilirostris.

References

¹ White, N., Sadeeshkumar, H., Sun, A. et al. Lithium-sensing riboswitch classes regulate expression of bacterial cation transporter genes. Sci Rep 12, 19145 (2022). https://doi.org/10.1038/s41598-022-20695-6
² England et. al. NanoLuc: A Small Luciferase is Brightening up the Field of Bioluminescence. Bioconjug Chem. 2016 May 18; 27(5): 1175–1187. doi:10.1021/acs.bioconjchem.6b00112.