DNA

Part:BBa_K2100049:Design

Designed by: Trinh Nguyen   Group: iGEM16_MIT   (2016-10-19)


pENTR attB-flipped eYFP-attP (TP901)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2163
    Illegal PstI site found at 1872
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2163
    Illegal NheI site found at 845
    Illegal NheI site found at 1111
    Illegal PstI site found at 1872
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2163
    Illegal BamHI site found at 1275
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2163
    Illegal PstI site found at 1872
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2163
    Illegal PstI site found at 1872
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 722


Design Notes

We used Golden Gate reaction with BsaI restriction enzyme to assembly the parts into the backbone vector. We don't want to have BsaI sites within the eYFP sequence. Additionally, we had to consider how to oriented the Q sites in the extended regions of the PCR primers, so that the eYFP gene would be upside-down after annealing into the backbone.


Source

Flipped eYFP was obtained from extension PCR from BBa_K2100005 (pENTR eYFP). The recognition sites of TP901 were ordered as single-single strands oligos from IDT and we got the sequences for the recognition sites from the BU Wetlab Team.

References