Part:BBa_K2916005:Design

LeuRS protein equipped with a 6x HIS affinity tag

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1104
  Illegal XhoI site found at 2581
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1071
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

The sequence was extracted from the pET21a-LeuRS-His plasmid and expressed in the BL21 (DE3) E.coli strain.

Codon optimization : 222 from G to C

Second stop codon was added in compliance with iGEM standards.

Source

Sebastian Maerkl Lab at EPFL, Switzerland.

http://www.addgene.org/124113/

References

McClain, William H. “Rules That Govern TRNA Identity in Protein Synthesis.” Journal of Molecular Biology, vol. 234, no. 2, Nov. 1993, pp. 257–80. DOI.org (Crossref), doi:10.1006/jmbi.1993.1582.

Cell-free translation reconstituted with purified components. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T. Nat Biotechnol. 2001 Aug;19(8):751-5. doi: 10.1038/90802. 10.1038/90802 PubMed 11479568

Lavickova, Barbora, and Sebastian J. Maerkl. A Simple, Robust, and Low-Cost Method to Produce the PURE Cell - Free System. preprint, Synthetic Biology, 18 Sept. 2018. DOI.org (Crossref), doi:10.1101/420570.