The pCAP03 plasmid has been successfully used to create capture vectors that can be used for TAR cloning of biosynthetic gene clusters. 'Capture arms' for a target BGC can be cloned within the restriction enzymes sites, and the plasmid contains elements that allow transfer between E. coli, actinobacteria and yeast cells.
- Apramycin resistance cassette (acc(3)IV)
- Neomycin and kanamycin resistance
- ΦC31 integrative site & integrase
- Origin of replication for Escherichia coli (pUC ori)
- ARSH4 autonomous replication sequence and CEN6 centromere for replication in yeast
- TRP1 marker for selection of yeast on tryptophan deficient media
- URA3 gene under the strong promoter pADH1 to select against non-homologous end joining in the presence of 5-fluoroorotic acid (5-FOA).
The plasmid was developed by Bradley Moore's lab as an upgraded version of pCAP011.
See AddGene link (here) to access the sequence.
The paper reporting pCAP03 is here
1. Yamanaka. K., Reynolds. K.A., Kersten. R.D., Ryan. K.S., Gonzalez. D.J., Nizet. V., Dorrestein. P.C., Moore. B.S. (2014) Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A. Proceedings of the National Academy of Sciences of the United States of America 111(5):1957-62. doi: 10.1073/pnas.1319584111