The mutate function designs the necessary set of primers for the desired mutations. An example is given in the vignette at https://github.com/ipb-halle/GoldenMutagenesis/blob/master/vignettes/Point_Mutagenesis.md
mutate_spm( input_sequence, prefix = "TT", restriction_enzyme = "GGTCTC", suffix = "A", vector = c("AATG", "AAGC"), replacements, replacement_range = 2, binding_min_length = 4, binding_max_length = 9, target_temp = 60, cuf = "e_coli_316407.csv", fragment_min_size = 100 )
The sequence which should be modified. This is an object of type character containing the sequence.
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
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: 2]
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
The Codon Usage Table which is being used to select the codon for an exchanged amino acid. [default: e_coli_316407.csv]
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 Point Mutation vignette and design the primers data(Point_Mutagenesis_BbsI_setup) primers<-mutate_spm(input_sequence, prefix="TT", restriction_enzyme = recognition_site_bbsi, suffix = "AA", vector=c("CTCA", "CTCG"), replacements = mutations, binding_min_length=4 , binding_max_length=9, target_temp=60, cuf=cuf)