The mutate_msd function designs the necessary set of primers for the desired mutations. Note that you can also select TGG in saturation mutagenesis to apply the 22c trick.
mutate_msd( input_sequence, codon = "NDT", prefix = "TT", restriction_enzyme = "GGTCTC", suffix = "A", vector = c("AATG", "AAGC"), replacements, replacement_range = 3, binding_min_length = 4, binding_max_length = 9, target_temp = 60, fragment_min_size = 100 )
The sequence which should be modified. This is an object of type character containing the sequence.
The desired type of MSD mutation [default: NDT]
Additional nucleobases in 5' position of the recognition site [default: TT]
Recognition site sequence of the respective restriction enzyme [default: GGTCTC]
Spacer nucleotides matching the cleavage pattern of the enzyme [default: A]
Four basepair overhangs complementary to the created overhangs in the acceptor vector [default: c("AATG", "AAGC")]
The desired substitutions. Can be a numeric vector or a list of character vectors with position and codon. See
Maximum distance in amino acid residues between two randomization sites to be incoporated into a single primer (reverse, end of the fragment) - has a cascading effect for following mutations [default: 3]
The minimal threshold value of the length of the template binding sequence in amino acid residues [default: 4]
Maximal length of the binding sequence in amino acid residues [default: 9]
Melting temperature of the binding sequence in
Minimal size of a generated gene fragment in base pairs [default 100]
An object of class Primerset with the designed Primers.
#Load the setup of the MSD vignette and design the primers data(MSD_BsaI_setup_lv2) print(mutations)#>  137 143 147 232 234print(recognition_site_bsai)#>  "GGTCTC"primers<-mutate_msd(input_sequence, prefix="TT" , restriction_enzyme=recognition_site_bsai, suffix="A", vector=c("AATG", "AAGC"), replacements=mutations, replacement_range=5, binding_min_length=4 , binding_max_length=9, target_temp=60, fragment_min_size=60 )