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Abstract:
Efficient assembly of large DNA constructs is a key technology in
synthetic biology. One of the most popular assembly systems is the MoClo
standard in which restriction and ligation of multiple fragments occurs
in a one-pot reaction. The system is based on a smart vector design and
type Its restriction enzymes, which cut outside their recognition site.
While the initial MoClo vectors had been developed for the assembly of
multiple transcription units of plants, some derivatives of the vectors
have been developed over the last years. Here we present a new set of
MoClo vectors for the assembly of fragment libraries and insertion of
constructs into bacterial chromosomes. The vectors are accompanied by a
computer program that generates a degenerate synthetic DNA sequence that
excludes "forbidden" DNA motifs. We demonstrate the usability of the new
approach by construction of a stable fluorescence repressor operator
system (FROS).
