The binary vectors contain Cas9 (nuclease or nickase) and the gRNA. Cas9 is codon optimized for high expression in monocots and dicots.
RNA is driven by the U6 promoter from Arabidopsis thaliana (for dicots) or Oryza sativa (for monocots).
Insertion of the target RNA in the Cas9 nickase vector can be easily done as DNA fragment containing both target RNAs at the ends. The inserted DNA fragment contains the U6 promoter.
Genes used in plant molecular biology sometimes have low expression in plants due to the fact that they have been originally isolated from bacteria which use a different codon usage. The betaglucuronidase (GUS) gene from E. coli was codon optimized for better expression in monocots and dicots. To prevent expression from bacteria the StLS1 intron was introduced. The synthetic GUS gene is regulated by the Ubi promoter from maize and introduced into maize embryos by particle gun bombardment. For comparison the GUS gene from Jefferson et al. (EMBO J. 87 Dec) was used, which is 100% identical in amino acid sequence and in the sourrounding of the genes.
For cloning of TALENs we have developed a binary vector which harbors both DNAs on one plasmid. Cloning of the TALE repeat domain can be done either in the SacI-BbvCI or into the AscI-HindIII restriction sites. The present vector contains the Ubi-maize promoter for transformation of monocots. But the TALENs cassette can be simply transfered with SfiI restriction to any other binary containing these restriction sites. Both backbones of the TALENs construct were codon optimized for high expression in monocots and dicots. They are identical in amino acid sequence. But to avoid problems due to repeated sequences, both backbones are not identical in the sequence of the nucleic acid. Both 35s promoters can be easily exchanged. We recommend small promoters, since cloning can be difficult, when the complete construct reaches 20 kb.
Introduction of different Kozak sequences for high, medium and low expression.
Optimized codon usage for monocots, dicots or plants.
In the section vectors you will get some information about the vectors we have already constructed. All the vectors, including the binaries, are sequenced. The list of vectors will grow continuously.
If the sequence of the vector and the DNA fragment is known, you will get the sequence and a map of the construct with the primers used for construction and the most important features. Cloning can be done in any vector you want.