Coding

Part:BBa_K3140002:Design

Designed by: Fahad Ali   Group: iGEM19_Sydney_Australia   (2019-10-12)
Revision as of 04:37, 15 October 2019 by Fahad3li (Talk | contribs) (→‎Source)


PsiK - 4-hydroxytryptamine kinase from Psilocybe cubensis


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 864
    Illegal SapI.rc site found at 973


Design Notes

This part was intended for use in pET-28c(+) for initial analysis of protein expression, and in pUS250 for final expression of a polycistronic construct incorporating PsiD, PsiK, and PsiM. To achieve this, the part was ordered in a gBlock containing BsaI sites (complementary to the backbone of pUS250, and to the BsaI site in the PsiK gBlock) for Golden Gate cloning, and with EcoRI and HindIII sites to enable traditional restriction cloning into pUS250. As there is a ribosomal binding site in the pET-28c(+) backbone, but not in pUS250, a RBS sequence was added to the gBlock upstream of the EcoRI site, so that it would not incorporate into pET-28c(+). The RBS sequence used was the consensus Shine-Dalgarno sequence.

Fig. 1: The gBlock incorporating part BBa_K3140002 (PsiK), a RBS, and sites for cloning into pET-28c(+) and pUS250.

Source

The native sequence for the 4-hydroxytryptamine kinase PsiK (Psilocybe cubensis) was obtained from NCBI: GenBank accession KY984099.1

References