Part:BBa_K2332040:Design
Blue light inducible expression of Intimin'-GFP-SpyTag (Pblind Intimin'-GFP-SpyTag)
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 2844
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 2844
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 2844
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 2844
Illegal NgoMIV site found at 880
Illegal NgoMIV site found at 1621 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2715
Design Notes
We decided to use Intimin' as this has been previously proven to function effectively for cell surface display of up to 30kDa passenger proteins (Wentzel et al., 2001). We included a linker between Intimin' and GFP and between GFP and SpyTag to facilitate mobility of GFP and SpyTag on the cell surface. The stop codon in GFP was removed for the generation of this fusion protein. Pblind promoter was designed by Jayaraman P. et al. (2016)
Source
Intimin protein sequence obtained from: http://www.uniprot.org/uniprot/P43261#sequences And reverse translated using: http://www.bioinformatics.org/sms2/rev_trans.html Intimin for the cell surface display of our tag was truncated according to Wentzel et al., 2001 SpyTag: BBa_K1159201 GFPmut 3b: Part:BBa_E0040