Part:BBa_K2244007:Design
ColE promoter +mhei gene +mcherry gene +T1terminator
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1250
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1250
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 754
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1250
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1250
Illegal AgeI site found at 413 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
LEV1 is a light repressor with an E. coli lexA DNA-binding domain, which binds to its cognate operator sequence on promoter when dimerized upon light irradiation. LEV1 is a fusion protein of VVD and LexA: VVD contains a light-oxygen-voltage (LOV) domain & N cap was added to the N-terminal of DNA-binding domain of LexA. The C-terminal domain of lexA repressor was removed. The expression of Lev1 is under a constitutive promoter so that it is constantly expressed.
ColE promoter, with an intrinsic RBS, is obtained from E. coli Col1 plasmid and contains a cognate operator sequence for LexA. Expression of target gene (in this case, mCherry) is under the control of ColE promoter, thus is also regulated by LEV1 binding and light irradiation. mCherry sequence is codon optimized for E. coli expression. T1 terminator is the most commonly used terminator in E. coli.