Part:BBa_K743010:Design
psb1C3_IntS recombination plasmid with RFP
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 1135
Illegal suffix found in sequence at 2226 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1135
Illegal SpeI site found at 2227
Illegal PstI site found at 2241
Illegal NotI site found at 1141
Illegal NotI site found at 2234 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1135
Illegal XhoI site found at 3689
Illegal XhoI site found at 4581 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1135
Illegal suffix found in sequence at 2227 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1135
Illegal XbaI site found at 1150
Illegal SpeI site found at 2227
Illegal PstI site found at 2241
Illegal NgoMIV site found at 867
Illegal AgeI site found at 1937
Illegal AgeI site found at 2049 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2783
Design Notes
The recombination sites are just 450bp long so that complete homology with any version of the CopS gene is achieved (i.e chromosomic and plasmidial versions) Though according to literature 1300 bp is the optimum size for recombination sites in Synechocystis, 450 bp yields reasonably high transformation efficiency.
Source
Recombination sites were PCR amplified from Synechocystis PCC6803 DNA. All the sequences between recombination sites are from psb4K5. The rest of the plasmid comes from psb1C3 sequence.
All the plasmid was built by Gibson assembly.
References
Giner-Lamia, J., Lopez-Maury, L., Reyes, J. C., & Florencio, F. J. (2012). The CopRS two-component system is responsible for resistance to copper in the cyanobacterium Synechocystis sp. PCC 6803. Plant physiology, 159(August), 1806-1818.