Part:BBa_K4046801:Design

hUBC - BS #1 - BS #1 - Kozak - cLuc - bghA

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1060
  Illegal BglII site found at 1119
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 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.

cLuc is an enzyme isolated from the genomic sequence of V. hilgendorfii , a marine crustacean. This gene was obtained through PCR of a commercially available plasmid pIND-CLucZ (Addgene, 53224). In normal function, cLuc is a luciferase enzyme that produces luminescence upon contact with its substrate, vargulin.

hUBC is a constitutive reporter that is associated with the ubiquitin c gene in humans. This gene was obtained through a FUGW-GFP plasmid, generously donated by the Gersbach lab at Duke University. In normal function, the hUBC 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