Reporter

Part:BBa_K3629025:Design

Designed by: Arshia Mostoufi   Group: iGEM20_Calgary   (2020-10-13)


mCherry expression construct with Gibson homology


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1620
    Illegal SpeI site found at 331
    Illegal PstI site found at 296
    Illegal PstI site found at 1015
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1620
    Illegal NheI site found at 121
    Illegal SpeI site found at 331
    Illegal PstI site found at 296
    Illegal PstI site found at 1015
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1620
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1620
    Illegal SpeI site found at 331
    Illegal PstI site found at 296
    Illegal PstI site found at 1015
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1620
    Illegal SpeI site found at 331
    Illegal PstI site found at 296
    Illegal PstI site found at 1015
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The construct is flanked with two Gibson homology sequences. Upon digestion with BbsI, the construct can be connected to either the LEU4 overproducing construct (BBa_K3629023) or the TRP2 overproducing construct (BBa_K3629024)on the 5' side and to the nourseothricin resistance expression construct (BBa_K3629015) on the 3' side. The Gibson homology sequences can also be used to easily add this fluorescent protein to any construct that contains the same homology sequences.

The construct was designed to be ultimately integrated into the genome of Yarrowia lipolytica. Therefore, a pair of gPCR primer binding sites flank the construct and can be used to help determine the location of the integration of the construct in the genome.

mCherry coding sequence was also codon-optimized for expression and function in Yarrowia lipolytica.

Source

The TEF intronic promoter (BBa_K3629001), and XPR2 terminator(BBa_K3629004) were both obtained from from genome sequence of Yarrowia lipolytica CDS: T01033.

mCherry (BBa_K3629021) coding sequence was obtained from https://www.fpbase.org/protein/mcherry/

References

1.[online] https://www.fpbase.org/protein/mcherry/ (Accessed October 26, 2020)

2.Blazeck, J., Liu, L., Redden, H., & Alper, H. (2011). Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Applied and environmental microbiology, 77(22), 7905–7914. https://doi.org/10.1128/AEM.05763-11