Coding
Part:BBa_K3075000:Design
Designed by: David Downes Group: iGEM19_UNSW_Australia (2019-10-07)
PAM-SnoopT-His
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 498
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 498
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 498
Illegal BamHI site found at 2131 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 498
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 498
Illegal NgoMIV site found at 393 - 1000COMPATIBLE WITH RFC[1000]
Design
The following gene construct was designed to enable the cloning and expression of the recombinant protein PAM-SnoopT-His within a T7 expression system.
Additions to the gene are as follows:
- Gibson forward and reverse overhangs – complementary overhangs outside the protein coding region enable cloning into pET19b plasmid via Gibson Assembly.
- Hexahistidine peptide tag – high affinity to Ni2+ - enables protein purification using Immobilised metal affinity chromatography (IMAC) via a Ni-NTA column.
Additionally, GSG linkers are included between the peptide sequences. This results in improved fluidity, allowing individual unhindered protein folding of each component (enzyme and tag system).
Figure 1: Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of PAM-SnoopT-His gene construct. Image by Linda Chen.
Source
Originated from Taxus wallichiana var. chinensis