Part:BBa_K3738023
Lbu-Cas13a with an N-terminal 6xHistidine Tag and C-Terminal Anionic Tag (Composite Part)
Cas13a is an enzyme originating from Leptotrichia buccalis (Lbu) which functions to cleave single-stranded RNAs (ssRNAs); particularly mRNAs. This function is achieved following protein-RNA complex formation with CRISPR RNA (crRNA) via crRNA backbone contacts with residues from the Helical-2, HEPN1, and Linker domains of Cas13a. The crRNA contains a spacer region coding for a direct repeat stem loop as well as a region complementary to target ssRNAs. Once the enzyme complex interacts with a target ssRNA, a structural conformation change occurs within the domains of the protein that permits active site formation for non-discriminate ssRNA cleavage (O’Connel et al., 2019).
This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, Lbu-Cas13a with an N-terminal 6xHistidine Tag and C-Terminal Anionic Tag coding region (BBa_K3738020), and double terminator BBa_B0015.The part is codon-optimized for use in E. coli It is improved from the Lethbridge High School iGEM 2019's Parts BBa_K3001003, BBa_K3001000 and BBa_K3001002 by introducing the anionic MS2 phage-like-particle uptake anionic tag as well as the transcriptional and translational regulators for optimal overexpression and 6XHistidine tag required for nickel affinity chromatography purification.
O'Connell MR. Molecular Mechanisms of RNA Targeting by Cas13-containing Type VI CRISPR–Cas Systems. Journal of Molecular Biology. 2019;431(1):66-87. doi: https://doi.org/10.1016/j.jmb.2018.06.029.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1134
Illegal PstI site found at 1989
Illegal PstI site found at 2928 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1134
Illegal PstI site found at 1989
Illegal PstI site found at 2928 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 415
Illegal BglII site found at 1351
Illegal BglII site found at 1561
Illegal BglII site found at 1615
Illegal BglII site found at 1846
Illegal BglII site found at 2119
Illegal BglII site found at 2605 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1134
Illegal PstI site found at 1989
Illegal PstI site found at 2928 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1134
Illegal PstI site found at 1989
Illegal PstI site found at 2928
Illegal NgoMIV site found at 2067
Illegal NgoMIV site found at 2703 - 1000COMPATIBLE WITH RFC[1000]
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