Difference between revisions of "Part:BBa K4046770:Design"
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===Design Notes===
 
===Design Notes===
−
This gene was synthesized commercially from the published sequence (IDT). tdTomato is the modified product of a gene originally from mushroom corals in the Discosoma family. 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).
+
This gene was synthesized commercially from the published sequence (IDT). tdTomato is the modified product of a gene originally from mushroom corals in the Discosoma family. 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). 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.
 +
 
  
  
Line 16: Line 17:
 
===Source===
 
===Source===
  
−
This is an identified binding site for the DhdR gene, and is generated from sequence analysis.
+
This is an 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.  
  
  

Revision as of 19:21, 20 October 2021


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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1025
    Illegal PstI site found at 1751
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1025
    Illegal PstI site found at 1751
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 614
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1025
    Illegal PstI site found at 1751
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1025
    Illegal PstI site found at 1751
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This gene was synthesized commercially from the published sequence (IDT). tdTomato is the modified product of a gene originally from mushroom corals in the Discosoma family. 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). 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.




Source

This is an 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

Sidney Cambridge (ed.), Photoswitching Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 1148, DOI 10.1007/978-1-4939-0470-9_7, © Springer Science+Business Media New York 2014