Part:BBa_K3075001:Design
DBATG38R/F301V-SnoopT-His
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 771
Illegal PstI site found at 826 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 771
Illegal PstI site found at 826 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 28
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 771
Illegal PstI site found at 826 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 771
Illegal PstI site found at 826 - 1000COMPATIBLE WITH RFC[1000]
Design
The following gene construct was designed to enable the recombinant expression of the DBATG38R/F301V protein within an E. coli chassis (Figure 3). Gibson forward and reverse overhangs were added the 5’ and 3’ ends for cloning via Gibson Assembly. The SnoopTag sequence was added to the C-terminal to enable the conjugation of the DBATG38R/F301V protein to SnoopCatcher containing beta-prefoldin arms of the assemblase scaffold. A hexa-histidine tag was added to the C-terminal end to enable the purification of the protein once expressed by immobilised metal affinity chromatography (IMAC) via a nickel-NTA column. Each gene component was separated by a GSG linker sequences to increase the flexibility between each peptide and prevent steric hindrance of protein folding.
The “Gibson fwd overhang - DBATG38R/F301V - GSG - SnoopTag - GSG -6XHisTag - Gibson reverse overhang” gBlock sequence (Figure 1) was codon optimised for E. coli chassis and synthesised as a single gene fragment by Integrated DNA Technologies (IDT).
Image
Figure 1: Sequence annotation of DBATG38R/F301V-SnoopT-His gBlock contains the DBAT gene (blue) with two mutations G38R and F301V (yellow), SnoopTag (pink) and a hexahistidine tag (grey) separated by GSG linkers (silver), TAA stop codon (brown) enclosed within Gibson forward and reverse overhangs (orange).
Source
Originated from Taxus cuspidata (Japanese yew)