Composite

Part:BBa_I4285:Design

Designed by: Austin Che   Group: Antiquity   (2004-10-02)


Trans-splicing Tetrahymena ribozyme to remove LVA tag


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 430
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 332
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The ribozyme was designed for trans-splicing based on the design principles in Kohler et al 1999. The target sequence is a EYFP gene with a LVA degradation tag. The target splice site is the U closest to the end of the EYFP. The IGS (P1 helix) of the ribozyme was redesigned to bind to this region and an extended antisense region to the LVA tag of around 50 bp was added. The spliced on 3' exon is simply a stop codon. The expected behavior is for the LVA tag to be replaced by a stop codon, allowing for a return to the normal degradation rate for the protein (i.e. long life) and thus higher expression.

Contains the same lac controlled promoter R0011 and double terminator used in the target construct.


Source

Tetrahymena thermophila

References

  • Kohler et al, JMB 1999 285(5):1935--1950 doi:10.1006/jmbi.1998.2447