Plasmid_Backbone

Part:BBa_K731700:Design

Designed by: Giacomo Giacomelli, Anna Depetris   Group: iGEM12_UNITN-Trento   (2012-08-14)
Revision as of 19:45, 23 August 2012 by Anna.d (Talk | contribs) (→‎Design Notes)

Platform for terminators analysis under the control of T7 promoter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 6850
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 6856
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 6850
    Illegal BglII site found at 6011
    Illegal BamHI site found at 6832
    Illegal XhoI site found at 753
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 6850
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 6850
    Plasmid lacks a suffix.
    Illegal XbaI site found at 6865
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 714
    Illegal NgoMIV site found at 1051
    Illegal NgoMIV site found at 4231
    Illegal NgoMIV site found at 4391
    Illegal NgoMIV site found at 5979
    Illegal AgeI site found at 6800
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 2228
    Illegal SapI.rc site found at 3310


Design Notes

When using and modifying this construct it is important to remember that the two proteins have strong N(C)-terminal homology. This should be taken into consideration when designing primers. The two proteins have partially overlapping emission spectra and different foldin kinetics.


For any future improvement, maybe it would be better two use two different fluorescence proteins, because these two has partially overlapping emission spectra and different folding kinetics. Using and modifying this construct is important to remember that the two proteins have strong N-terminal homology, especially designing primers.

Source

The part was built starting from a construct kindly provided from the Mansy lab made by Roberta Lentini(RL024). The construct is a pET21b where mCherry and A206K Venus were inserted with a 20 bp spacer between the 2 proteins. That plasmid was mutated two times to eliminate illegal restriction sites and a prefix-suffix linker was added by mutagenesis.

References