Plasmid
pEX

Part:BBa_K243033:Design

Designed by: Freiburg Bioware09   Group: iGEM09_Freiburg_bioware   (2009-10-20)

pEX


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal suffix found in sequence at 2734
    Illegal XbaI site found at 1523
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 2735
    Illegal PstI site found at 2749
    Illegal NotI site found at 2742
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1808
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal suffix found in sequence at 2735
    Illegal XbaI site found at 1523
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal suffix found in sequence at 2725
    Illegal XbaI site found at 1523
    Illegal NgoMIV site found at 1531
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3800
    Illegal SapI.rc site found at 4799


Design Notes

Commented GenBank file

As we needed a suitable expression vector, we altered the NEB vector pMAL-p5x according to our needs. Via PCR the chloramphenicol acetyle transferase (CAT) was amplified out of a lab vector. Add-on-tail primers were used for the PCR containing the prefix XbaI and NgoMIV and the suffix AgeI, SpeI, NotI and PstI. EcoRI and NotI were excluded so that the ribosome binding site (RBS) stays within a distance of 7 nucleotides from the start codon. The add-on-tail primers also included the RBS GAAA ressembling the highly efficient shine dalgarno sequence of Biobrick part: BBa_B0030. In front of the prefix MfeI and behind the suffix HindIII were inserted to enable the exchange of the MalE gene with the CAT gene.

Source

By adding additional restriction sites in pMAL we create our expression vector.

References