Part:BBa_K243033:Design
pEX
- 10INCOMPATIBLE WITH RFC[10]Illegal suffix found in sequence at 2734
Illegal XbaI site found at 1523 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 2735
Illegal PstI site found at 2749
Illegal NotI site found at 2742 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1808
- 23INCOMPATIBLE WITH RFC[23]Illegal suffix found in sequence at 2735
Illegal XbaI site found at 1523 - 25INCOMPATIBLE WITH RFC[25]Illegal suffix found in sequence at 2725
Illegal XbaI site found at 1523
Illegal NgoMIV site found at 1531 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 3800
Illegal SapI.rc site found at 4799
Design Notes
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.