Part:BBa_K4046990:Design

CMV - BS #2 with spacer - BS #2 with spacer - BS #2 - Kozak - mCherry - bghA

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1065
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1065
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 614
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1065
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1065
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

dhdO sequences were designed and optimized for binding to the DhdR protein. Furthermore, a promoter, Kozak sequence, and a terminator were added.

Source

This includes identified binding site for the DhdR gene, as described by Xiao et al, 2021. This gene was synthesized commercially from the published sequence (IDT). The DhdR gene is a transcriptional repression factor that is derived from the bacteria Achromobacter denitrificans. The dhdO binding sites were designed and modified to improve binding behavior of the DhdR protein to the sequence.

mCherry is a fluorescent gene product derived from the DsRed gene of mushrooms corals in the Discosoma family. This gene was obtained directly through assembly into the pcDNA5 plasmid (Thermo Fischer, V103320), which contained the mCherry gene. In normal function, mCherry is a red fluorescence protein.

CMV is a constitutive reporter associated with the cytomegalovirus. This gene was obtained through the pcDNA5 backbone that we were using (Thermo Fischer, V103320). In normal function, the CMV promoter allows for high levels of expression of associated gene products.

References

Xiao, D., Zhang, W., Guo, X., Liu, Y., Hu, C., Guo, S., Kang, Z., Xu, X., Ma, C., Gao, C., & Xu, P. (2021). A D-2-hydroxyglutarate biosensor based on specific transcriptional regulator DHDR. https://doi.org/10.1101/2021.02.18.430539

Cambridge, S. (2014). Photoswitching proteins: Methods and protocols (Vol. 1148). Humana Press.