Part:BBa_K3075000:Design

PAM-SnoopT-His

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 498
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 498
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 498
  Illegal BamHI site found at 2131
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 498
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 498
  Illegal NgoMIV site found at 393
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design

The following gene construct was designed to enable the cloning and expression of the recombinant proteins PAM-SnoopT and TycA-SpyT 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 and TycA-SpyT gene constructs. Image by Linda Chen.

Source

Originated from Taxus wallichiana var. chinensis