Difference between revisions of "Part:BBa K3407005:Design"
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Revision as of 18:32, 26 October 2020
Fox-1 RBD* : a mutated version of Fox-1 RBD with depleted binding capacity
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Fox-1 amino acid sequence retrieved from NCBI residues 109–208 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, and mutations described as deleterious for its binding capacity [1] were added manually to the gene sequence (mutations F160A and F126A). The Cry7Ca1 gene 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, XbaI, SpeI, PstI) and Type IIS forbidden restriction sites (BsaI, SapI and Bpil). BglBrick prefix (nt 1525-1560) and suffix (nt 2277-2318) from pBbB7a (Addgene #35358) were added to the optimised sequence generated. They were designed to be cloned with Gibson Assembly.
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].
References
- 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.
- 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