Plasmid

Part:BBa_K090403:Design

Designed by: Daniel Goodman   Group: iGEM08_Cambridge   (2008-10-29)


Gram-positive Shuttle Vector for Chromosomal Integration


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 5611
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 5611
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 5617
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 5611
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 5611
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 5611
    Illegal XbaI site found at 5626
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 1189
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3984


Design Notes

We attempted to design the PCR ends to not interfere with the AmyE flanking regions, and also to have the same annealing temperatures. To see the primers we used, see our Primer page under the Bacillus subproject: [http://2008.igem.org/Team:Cambridge/Bacillus/Primers]


Source

As explained in the image, this vector was assembled at once from 4 separate pieces. The green and red regions (biobrick cut site and E. coli replication/Amp resistance) come from PBS1A3. The blue pieces are the flanking AmyE regions from ECE112, which was obtained via the Bacillus Genetic Stock Center.

References