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
)

## Arguments

input_sequence 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 vignette("MSD3", package = "goldenmutagenesis") for examples. 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 print('\u00B0')C [default: 60] Minimal size of a generated gene fragment in base pairs [default 100]

## Value

An object of class Primerset with the designed Primers.

## Examples

#Load the setup of the MSD vignette and design the primers
data(MSD_BsaI_setup_lv2)
print(mutations)#> [1] 137 143 147 232 234print(recognition_site_bsai)#> [1] "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 )