Difference between revisions of "Part:BBa K1723005:Design"
E.Cuillery (Talk | contribs) (→Design Notes) |
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===Design Notes=== | ===Design Notes=== | ||
| − | This part was synthesized on the model of the PAM rich URS J23117 promoter (BBa_K1723001). The promoter BBa_K1723001 was mutated to change the sequences of the sgRNA-targeted sites therefore allowing the presence of two independently regulated promoters. We avoided mutating -35 sequence and -10 sequence to keep a similar promoter strength [1]. the new targeted sequences were generated randomly and blasted on ''E. coli'' genome to avoid unwanted interactions. Using this procedure we believe more similar promoters could be made. | + | This part was synthesized on the model of the PAM rich URS J23117 promoter (BBa_K1723001). The promoter BBa_K1723001 was mutated to change the sequences of the sgRNA-targeted sites therefore allowing the presence of two independently regulated promoters. We avoided mutating -35 sequence and -10 sequence to keep a similar promoter strength [1]. the new targeted sequences were generated randomly and blasted on ''E. coli'' genome to avoid unwanted interactions. Using this procedure we believe more similar promoters could be made. In our experiments the promoter is 40bp upstream of the ATG start codon. |
===Source=== | ===Source=== | ||
Revision as of 21:55, 17 September 2015
PAM rich URS J23117Alt promoter
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 277
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 167
Illegal XhoI site found at 195 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
This part was synthesized on the model of the PAM rich URS J23117 promoter (BBa_K1723001). The promoter BBa_K1723001 was mutated to change the sequences of the sgRNA-targeted sites therefore allowing the presence of two independently regulated promoters. We avoided mutating -35 sequence and -10 sequence to keep a similar promoter strength [1]. the new targeted sequences were generated randomly and blasted on E. coli genome to avoid unwanted interactions. Using this procedure we believe more similar promoters could be made. In our experiments the promoter is 40bp upstream of the ATG start codon.
Source
this part was fully synthesized.
References
[1] Bikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., & Marraffini, L. A. (2013). Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic acids research, 41(15), 7429-7437.
[2] Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173-1183.
[3] Alec AK Nielsen & Christopher A Voigt (2014). Multi-input CRISPR/Cas circuits that interface host regulatory network. Molecular systems biology, 10(11), 763.