Composite

Part:BBa_K3237017

Designed by: Aubrey Demchuk, Kristi Turton   Group: iGEM19_Lethbridge   (2019-10-11)


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

This part is for the expression of insulin in microalgae, specifically Chlamydomonas reinhadtii . In its design there are the following synthetic components. The proinsulin part of the construct has been modified in that the original protease cut sites in proinsulin has been replaced with the Furin protease cut site. Furin is a also a human protein that will still allow for protein processing in an active form [1]. The second component is that this proinsulin is fused with red fluorescent protein (mRFP1) with a furin cut site and linker in between for removal. The third component is the addition of a his tag on the N-terminus to allow for purification from microalgae if necessary (although not proposed as a part of our project system). In summary the part has a his tag on the N-terminus, which is attached to the mRFp1-proinsulin fusion. This fusion can be broken with the introduction of furin protease, and proinsulin will be processed simultaneously with the furin addition as well.

This part is codon optimized for use in C. reinhardtti but has a construct counterpart codon optimized for E.coli , BBa_K3237002. This part is also an improved part from Tsinhua 2014.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1783
    Illegal XbaI site found at 1771
    Illegal XbaI site found at 1813
    Illegal SpeI site found at 1807
    Illegal PstI site found at 1321
    Illegal PstI site found at 1789
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1783
    Illegal NheI site found at 1335
    Illegal SpeI site found at 1807
    Illegal PstI site found at 1321
    Illegal PstI site found at 1789
    Illegal NotI site found at 1819
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1783
    Illegal BamHI site found at 1801
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1783
    Illegal XbaI site found at 1771
    Illegal XbaI site found at 1813
    Illegal SpeI site found at 1807
    Illegal PstI site found at 1321
    Illegal PstI site found at 1789
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1783
    Illegal XbaI site found at 1771
    Illegal XbaI site found at 1813
    Illegal SpeI site found at 1807
    Illegal PstI site found at 1321
    Illegal PstI site found at 1789
    Illegal AgeI site found at 881
    Illegal AgeI site found at 993
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None