Part:BBa_K3407020:Design

Fox-1 RBD domain binding RNA sequence UGCAUGU with T7 promoter - RBS - T1 terminator Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 55
  Illegal BamHI site found at 444
  Illegal XhoI site found at 453
  
• 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]. 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