Part:BBa_K4046799:Design
hUBC - BS #1 - Kozak - tdTomato - BghA
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 780
Illegal PstI site found at 1506 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 780
Illegal PstI site found at 1506 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 780
Illegal PstI site found at 1506 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 780
Illegal PstI site found at 1506 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
dhdO sequences were designed and optimized for binding to the DhdR protein. Furthermore, a promoter, Kozak sequence, 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.
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