Plasmid
pBSEP1

Part:BBa_K090401:Design

Designed by: Daniel Goodman   Group: iGEM08_Cambridge   (2008-10-27)


Gram-positive Shuttle Vector for Episomal Expression


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 5187
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 5187
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 5193
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 5187
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 5187
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 5187
    Illegal XbaI site found at 5202
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1533
    Illegal BsaI.rc site found at 4261
    Illegal SapI site found at 3178


Design Notes

We had to remove a PstI site inside the coding region of the beta-lactamase gene coding for Ampicillin resistance, and thus the backbone had to be PCRed from two parts, with a joining primer filling in cut site with a degenerate codon.


Source

This part was made via InFusion assembly from many separate pieces. The backbone comes from PAD123 (Bacillus Stock Center catalog number ECE166), and contains replication origins for B. subtilis and E. coli, as well as resistance markers for Ampicillin and Chloramphenicol.

References