Part:BBa_K2319009:Design

6xHis-mCherry under T7 expression system

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 768
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

HindIII, ATG, AgeI

A HindIII restriction site, a start codon and an AgeI restriction site are added immediately downstream of the T7 promoter+RBS. A number of design considerations motivated the choice of these restriction sites. The HindIII site (A\AGCTT) — sandwiched between the RBS and the start codon — has a sequence very similar to the optimal sequence predicted by the sequence logo of E. coli ribosome binding sites, which can be found here. In fact, the HindIII sequence is closer to the optimal sequence than the typical 5'-TACTAG-3' mixed SpeI-XbaI restriction site formed by BioBrick assembly, improving the initial ribosome-mRNA binding and improves translation.

The AgeI site was chosen to simplify assembly of fusion proteins in this backbone: by inserting a protein coding sequence at the N-terminus of the existing protein using the HindIII and AgeI sites, a fusion protein can be formed with a benign scar. The AgeI site (A\CCGGT) is translated in-frame to Thr-Gly, amino acids commonly used in linker sequences for fusion proteins. Threonine's hydroxyl group makes it hydrophilic — allowing stabilizing interactions with the aqueous cellular environment — while glycine's small size makes the linker more flexible, allowing both protein domains to fold independently. In addition, the AgeI site is useful if the user wishes to transfer an RFC25-compatible fusion protein (Freiburg format) into our expression system.

6xHis tag

The His tag was codon-optimized for this protein.

Source

This part was designed using existing BioBricks and some primers to add overhangs.