Part:BBa_K3407005:Design

Fox-1 RBD* : a mutated version of Fox-1 RBD with depleted binding capacity

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Fox-1 amino acid sequence was retrieved from NCBI, and residues 109–208 corresponding to the RNA Binding Domain (RBD) were used following the Literature’s materials and methods [1] and mutations F126A and F160A were incorporated to the amino acid sequence. As an N-HisTag from pET-28(a) was used in materials and methods, its amino acid sequence was added in the N-Terminal of Fox-1 RBD. The Fox-1 RBS sequence was codon-optimized for expression in Escherichia coli using the GenSmartTM Codon Optimization Tool. The forbidden restriction enzymes sites were the following: BioBrick forbidden restriction sites (EcoRI, BglII, XhoI, BamHI). They were designed to be cloned with Gibson Assembly into pBbB7a backbone.

Source

Plasmids used were from BglBrick repository. pBbB7a-GFP was a gift from Jay Keasling (Addgene plasmid # 35358; http://n2t.net/addgene:35358 ; RRID:Addgene_35358) [2]. Please refer to the Addgene page for more information about licences associated with the use of the plasmid.

References

1. Auweter, S., Fasan, R., Reymond, L., Underwood, J., Black, D., Pitsch, S. and Allain, F., 2020. Molecular Basis Of RNA Recognition By The Human Alternative Splicing Factor Fox-1.
2. BglBrick vectors and datasheets: A synthetic biology platform for gene expression. Lee TS, Krupa RA, Zhang F, Hajimorad M, Holtz WJ, Prasad N, Lee SK, Keasling JD. J Biol Eng. 2011 Sep 20;5:12. 10.1186/1754-1611-5-12 PubMed 21933410