Composite

Part:BBa_K3237019:Design

Designed by: Aubrey Demchuk, Kristi Turton   Group: iGEM19_Lethbridge   (2019-10-14)


His-mRFP1-SCI57 with furin cut sites codon optimized for C. reinhardtii


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1700
    Illegal XbaI site found at 1688
    Illegal XbaI site found at 1730
    Illegal SpeI site found at 1724
    Illegal PstI site found at 1238
    Illegal PstI site found at 1706
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1700
    Illegal NheI site found at 1252
    Illegal SpeI site found at 1724
    Illegal PstI site found at 1238
    Illegal PstI site found at 1706
    Illegal NotI site found at 1736
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1700
    Illegal BamHI site found at 1718
    Illegal XhoI site found at 656
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1700
    Illegal XbaI site found at 1688
    Illegal XbaI site found at 1730
    Illegal SpeI site found at 1724
    Illegal PstI site found at 1238
    Illegal PstI site found at 1706
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1700
    Illegal XbaI site found at 1688
    Illegal XbaI site found at 1730
    Illegal SpeI site found at 1724
    Illegal PstI site found at 1238
    Illegal PstI site found at 1706
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The linkers were added after the furin cut sites to allow for space for the protease to be able to cut the amino acid sequence and to prevent steric hinderance.


Source

The part coding sequence was found from the literature [3] and the RFP sequence was taken from the registry BBa_E1010. The part was synthesized by IDT.


References

1. Hua,Q. (2010) Insulin: a small protein with a long journey. Protein Cell. 6, 537-551.


2. 1. Hay, C., and Docherty, K. (2003) Enhanced expression of a furin-cleavable proinsulin. Journal of molecular endocrinology. 3, 597-607.


3. Hua, Q., Nakagawa, S., Jia, W., Huang, K., Philips, N., Hu, S., and Wiess, M. (2008) Design of an active ultrastable single-chain insulin analog: synthesis, structure, and therapuetic implications. The Journal of Biological Chemistry. 21, 1473-14716.