Part:BBa_K3114006:Design
Water-soluble chlorophyll binding protein (6GIX) with 6xHis tag
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 594
Illegal XhoI site found at 4 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 119
Illegal AgeI site found at 62
Illegal AgeI site found at 378 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1
Illegal BsaI.rc site found at 605
Design Notes
When designing this part and the rest of our collection, we were interested in creating parts that could be used in Golden Gate assembly right out of the distribution kit without the need to first domesticate them in a Golden Gate entry vector. As such, these parts are not compatible with the iGEM Type IIS RFC[1000] assembly standard because we included the BsaI restriction site and MoClo standard fusion site in the part’s sequence.
As per the MoClo standard, the 5’ cds fusion sequence included in this part is AGGT, and the 3’ cds fusion sequence is GCTT (Weber et al., 2011).
This part does not contain a start codon, as it was designed to be used with one of the signal peptides in the collection. The native L. virginicum signal peptide was excluded from this sequence. A double stop codon was introduced to the sequence.
A 6X Histidine affinity chromatography tag was added to the N-terminus of this sequence for purification. Our modelling informed us that this tag would likely not interfere with 6GIX’s folding or function. Regardless, we added a thrombin proteolytic site between the tag and the 6GIX sequence in case it needed to be removed following purification.
The sequence has been codon optimized for high expression in E. coli.
Source
This part was synthesized. The sequence was originally obtained from PDB.
References
Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A modular cloning system for standardized assembly of multigene constructs. PLoS One. doi: 10.1371/journal.pone.0016765