Generator
KanMX(lox)

Part:BBa_K300989:Design

Designed by: Lorenzo Pasotti, Giacomo Zambianchi, Federica Sampietro, Paolo Magni   Group: iGEM10_UNIPV-Pavia   (2010-10-03)

[LoxP-KanMX-LoxP] excisable marker for yeast


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 39
    Illegal PstI site found at 254
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 254
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 44
    Illegal XhoI site found at 1492
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 39
    Illegal PstI site found at 254
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 39
    Illegal PstI site found at 254
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The original sequence of loxP-KanMX-loxP from pUG6 vector has two NsiI restriction sites in kanR(Tn903) coding sequence and they have been removed in this part by changing codons.

The TEF2 promoter has a XbaI and a PstI restriction sites, which have not been removed to avoid the change of the promoter strength. For this reason, this part is not fully compatible with RFC10 BioBrick Standard Assembly, but it can be assembled by using EcoRI-SpeI enzymes.


Source

pUG6 vector (GenBank: AF298793.1).

References

[1] Guldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996), A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Research, Vol. 24, No. 13 2519–2524.

[2] Gueldner U, Heinisch J, Koehler GJ, Voss D, Hegemann JH (2002), A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Research, Vol. 30, No. 6 e23.