Part:BBa_K4654019:Design
T7-Promoter_Spacer1-Li-+II_Riboswitch-Spacer2-5'nhaA_mScarlet-I3
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 30
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 30
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 30
Illegal BglII site found at 878 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 30
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 30
Illegal NgoMIV site found at 123
Illegal NgoMIV site found at 336 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
During the design process we made sure to include the spacer sequences up- and downstream of the riboswitch because they influence the folding of the riboswitch. We originally did not know where the RBS was located in Spacer Region 2 so we had to verify the location by asking the authors. We codon optimized the mScarlet-I3 sequence for expression in E. coli.
Source
The T7 promoter is derived from the T7 phage. The riboswitch can be found in certain bacteria, the sequence we are using was created by White et. al. by creating a consenus sequence from different sequences of the riboswtich family1. mScarlet-I3 belongs to the family of red fluorescent proteins and was not isolated from an organism but was derived from a synthetic construct, originally derived from mCherry.2
References
1 White, N., Sadeeshkumar, H., Sun, A. et al. Lithium-sensing riboswitch classes regulate expression of bacterial cation transporter genes. Sci Rep 12, 19145 (2022). https://doi.org/10.1038/s41598-022-20695-6
2 Gadella Twj, Van Weeren L, Stouthamer J, Hink Ma, Wolters Ahg, Giepmans Bng, Aumonier S, Dupuy J, Royant A (2023). Nature Methods, , . doi: 10.1038/s41592-023-01809-y.