Difference between revisions of "Part:BBa K3407024:Design"
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<partinfo>BBa_K3407024 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3407024 SequenceAndFeatures</partinfo> | ||
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===Design Notes=== | ===Design Notes=== | ||
− | Fox-1 amino acid sequence retrieved from NCBI residues 109–208 following the Literature’s materials and methods <html><a href="#1">[1]</a></html>. 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 | + | 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 <html><a href="#1">[1]</a></html> 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 <i>Escherichia coli</i> 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. |
Latest revision as of 03:11, 28 October 2020
Fox-1 RBD* domain with depleted binding capacity, with T7 promoter - RBS - T1 terminator
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 55
Illegal BamHI site found at 444
Illegal XhoI site found at 453 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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
- 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