Composite

Part:BBa_K3738023

Designed by: Emily Hagens and Mark Lea   Group: iGEM21_Lethbridge   (2021-10-20)
Revision as of 17:06, 20 October 2021 by Marklea (Talk | contribs)


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.

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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1134
    Illegal PstI site found at 1989
    Illegal PstI site found at 2928
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1134
    Illegal PstI site found at 1989
    Illegal PstI site found at 2928
  • 21
    INCOMPATIBLE 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
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1134
    Illegal PstI site found at 1989
    Illegal PstI site found at 2928
  • 25
    INCOMPATIBLE 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
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None