Part:BBa_K4046700:Design

CMV - BS #1 - Kozak - tdTomato - bghA

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 998
  Illegal PstI site found at 1724
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 998
  Illegal PstI site found at 1724
  
• 21
  INCOMPATIBLE WITH RFC[21]

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

  Illegal PstI site found at 998
  Illegal PstI site found at 1724
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 998
  Illegal PstI site found at 1724
  
• 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 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.

tdTomato is the modified product of a gene originally from mushroom corals in the Discosoma family. The gene was obtained through PCR of a commercially available plasmid pCRISPR-HOT_tdTomato (Addgene, 138567). In normal function, tdTomato 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

A D-2-hydroxyglutarate biosensor based on specific transcriptional regulator DhdR Dan Xiao, Wen Zhang, Xiaoting Guo, Yidong Liu, Chunxia Hu, Shiting Guo, Zhaoqi Kang, Xianzhi Xu, Cuiqing Ma, Chao Gao, Ping Xu bioRxiv 2021.02.18.430539; doi: https://doi.org/10.1101/2021.02.18.430539