Part:BBa_K2649001:Design
cpComE-NLS-VP16-NES SM6
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 4
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1
Design Notes
We fused a human-codon optimized cpComE coding sequence to a nuclear localization sequence, a VP16 activating domain, and a nuclear export sequence, with a goal to adapt its function to mammalian expression and signalling. ComE's native S. mutans do not have a nucleus, so we hope that the NLS and NES would assist in shuttling of cpComE back and forth through the nuclear envolope. Additionally, we decided to fuse VP16 to cpComE, to ensure that it would activate transcription in mammalian cells, if it binds to DNA.
Source
The ComE protein is from the Streptoccocus mutans strain SM6. The protein sequence is item A0A0E2EP61 in the UniprotKB database, and the corresponding codon sequence was optimized for human expression. D60E mutations were previously used as a phosphomimetic control for ComE in "Oligomerization of the Response Regulator ComE from Streptococcus mutans Is Affected by Phosphorylation" (Goodman et al., 2012. DOI: 10.1128/JB.06565-11) The NLS, VP16, and NES sequences were taken from Systematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells (Weiss and Voigt et al., 2014. DOI: 10.1021/sb5002856)