Composite

Part:BBa_K2207014:Design

Designed by: Junming Qian   Group: iGEM17_ZJU-China   (2017-10-25)


PhlAC enzyme


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 4607
    Illegal PstI site found at 776
    Illegal PstI site found at 4619
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 776
    Illegal PstI site found at 4619
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1494
    Illegal XhoI site found at 2168
    Illegal XhoI site found at 3079
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 4607
    Illegal PstI site found at 776
    Illegal PstI site found at 4619
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 4607
    Illegal PstI site found at 776
    Illegal PstI site found at 4619
    Illegal NgoMIV site found at 1298
    Illegal NgoMIV site found at 1581
    Illegal NgoMIV site found at 1868
    Illegal AgeI site found at 3388
    Illegal AgeI site found at 3416
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 486
    Illegal SapI site found at 168


Design Notes

We used the F2A sequenece（gtgaaacagactttgaattttgaccttctcaagttggcgggagacgtggagtccaaccctggacct）to link the phlA with phlC, and then combined them to eGFP to construct a fusion protein so that the two genes can be transcribed as a single mRNA and generating two independent gene products, and meanwhile we can identify whether the proteins are expressed by detecting the fluorescent.

Source

We successfully cloned the phlAC via colony PCR from Pseudomonas fluorescens 2P24, which is a gift from Prof. Liqun Zhang of China Agriculture University.

References

Jin H K, Lee S R, Li L H, et al. High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice[J]. Plos One, 2011, 6(4):e18556.