Multiple Site Saturation Mutagenesis Example 2

Chris Ulpinnis & Pascal Püllmann

2020-08-28

Multiple Site Saturation Mutagenesis

You can find the lastest version of this file at https://github.com/ipb-halle/GoldenMutagenesis/blob/master/vignettes/MSD2.md

Experimental Workflow

Target sequence

open reading frame YfeX in pCA24N (Chloramphenicol resistance)

Clone into

seperate gene fragments into pAGM9121 first then reassemble into pAGM22082_cRed

Genomic sequence YfeX

ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGGGAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACGCGCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCAGAAGAGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACGACCCAGTTCGATGTGTTGATCCACATTCTTTCTCTGCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTTGAAGAGCGTGACCTGAGCGGCTTTGTTGACGGTACGGAAAACCCGGCGGGTGAAGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCGGGCGGCAGCTATGTGTTTGTCCAGCGTTGGGAACACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGGTGATCGGGCGCACCAAAGAGGCCAACGAAGAGATCGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTCGCCAGAGCCTGCCGTACGGCACTGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACTGCGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA

Restriction Enzyme

Level 0

BbsI

Recognition site: GAAGAC

Level 2

BsaI

Recognition site: GGTCTC

Envisioned Mutations

Alanine - 51
Leucine - 101
Glutamic Acid - 151
Asparagine - 202
Cysteine - 252

Substitute for NDT

R Workflow

library("GoldenMutagenesis")

## Loading required package: seqinr

## Loading required package: stringr

input_sequence<-"ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGGGAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACGCGCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCAGAAGAGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACGACCCAGTTCGATGTGTTGATCCACATTCTTTCTCTGCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTTGAAGAGCGTGACCTGAGCGGCTTTGTTGACGGTACGGAAAACCCGGCGGGTGAAGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCGGGCGGCAGCTATGTGTTTGTCCAGCGTTGGGAACACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGGTGATCGGGCGCACCAAAGAGGCCAACGAAGAGATCGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTCGCCAGAGCCTGCCGTACGGCACTGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACTGCGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA"
recognition_site_bbsi<-"GAAGAC"
recognition_site_bsai<-"GGTCTC"
cuf<-"e_coli_316407.csv"

The domesticate function checks for internal cleavage sites. If corresponding sites are present silent mutations are introduced to destroy the recognition sites. The functions returns a list containing the position of the choosen amino acid residue for silent mutation.

mutations_bbsi<-domesticate(input_sequence, recognition_site_bbsi, cuf=cuf)

## No domestication needed.

mutations_bbsi

## list()

mutations_bsai<-domesticate(input_sequence, recognition_site_bsai, cuf=cuf)

## No domestication needed.

mutations_bsai

## list()

The mutate_msd function designs the necessary set of primers for the desired mutations. 
The function has the following parameters:
prefix: Additional nucleobases in 5’ position of the recognition site [default: TT]
restriction_enzym: Recognition site sequence of the respective restriction enzyme [default: GGTCTC]
codon: The codon which should be used in the mutagenesis [default: NDT]
suffix: Spacer nucleotides matching the cleavage pattern of the enzyme [default: A]
vector: Four basepair overhangs complementary to the created overhangs in the acceptor vector [default: c(“AATG”, “AAGC”)]
replacements: The desired substitutions as a vector with positions OR a list containing vetors with position (char) and type of MSD mutation (char) (see MSD3.rd for an example)
binding_min_length: The minimal threshold value of the template binding sequence in amino acid residues [default: 4]
binding_max_length: Maximal length of the binding sequence [default: 9]
target_temp: Melting temperature of the binding sequence in °C [default: 60]
replacement_range: Maximum distance between two randomization sites to be incoporated into a single primer in amino acid residues [default: 5]
fragment_min_size: Minimal size of a generated gene fragment in base pairs [default 100]

It will return an object of the class Primerset.
The primers for multiple site saturation mutagenesis have an additional slot called “NDT”. This slot contains a non-binding region in which (the) NDT site(s) is/are located.

#If domestication is necessary follow the workflow of the Point Mutagenesis vignette
mutations<-c(51,101,151,202,252)
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 )
primers

## An object of class "Extended_Primerset"
## Slot "fragments":
## [[1]]
## An object of class "Fragment"
## Slot "start":
## [1] 2
## 
## Slot "stop":
## [1] 53
## 
## Slot "start_mutation":
## logical(0)
## 
## Slot "stop_mutation":
## [1] 51
## 
## 
## [[2]]
## An object of class "Fragment"
## Slot "start":
## [1] 54
## 
## Slot "stop":
## [1] 103
## 
## Slot "start_mutation":
## logical(0)
## 
## Slot "stop_mutation":
## [1] 101
## 
## 
## [[3]]
## An object of class "Fragment"
## Slot "start":
## [1] 104
## 
## Slot "stop":
## [1] 153
## 
## Slot "start_mutation":
## logical(0)
## 
## Slot "stop_mutation":
## [1] 151
## 
## 
## [[4]]
## An object of class "Fragment"
## Slot "start":
## [1] 154
## 
## Slot "stop":
## [1] 204
## 
## Slot "start_mutation":
## logical(0)
## 
## Slot "stop_mutation":
## [1] 202
## 
## 
## [[5]]
## An object of class "Fragment"
## Slot "start":
## [1] 205
## 
## Slot "stop":
## [1] 254
## 
## Slot "start_mutation":
## logical(0)
## 
## Slot "stop_mutation":
## [1] 252
## 
## 
## [[6]]
## An object of class "Fragment"
## Slot "start":
## [1] 255
## 
## Slot "stop":
## [1] 300
## 
## Slot "start_mutation":
## logical(0)
## 
## Slot "stop_mutation":
## logical(0)
## 
## 
## 
## Slot "oldsequence":
## [1] "ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGGGAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACGCGCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCAGAAGAGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACGACCCAGTTCGATGTGTTGATCCACATTCTTTCTCTGCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTTGAAGAGCGTGACCTGAGCGGCTTTGTTGACGGTACGGAAAACCCGGCGGGTGAAGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCGGGCGGCAGCTATGTGTTTGTCCAGCGTTGGGAACACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGGTGATCGGGCGCACCAAAGAGGCCAACGAAGAGATCGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTCGCCAGAGCCTGCCGTACGGCACTGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACTGCGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA"
## 
## Slot "primers":
## [[1]]
## [[1]][[1]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GGTCTC"
## 
## Slot "suffix":
## [1] "A"
## 
## Slot "vector":
## [1] "AATG"
## 
## Slot "overhang":
## [1] ""
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "TCTCAGGTTCAGAGTGGCATTTTGCC"
## 
## Slot "temperature":
## [1] 60.36446
## 
## Slot "difference":
## [1] 0.3644563
## 
## 
## [[1]][[2]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GGTCTC"
## 
## Slot "suffix":
## [1] "T"
## 
## Slot "vector":
## [1] ""
## 
## Slot "overhang":
## [1] "AAGA"
## 
## Slot "extra":
## [1] "TGAHN"
## 
## Slot "binding_sequence":
## [1] "GTCCGGGAATTTCGCTTCAAAAGTTGC"
## 
## Slot "temperature":
## [1] 60.56425
## 
## Slot "difference":
## [1] 0.1997931
## 
## 
## 
## [[2]]
## [[2]][[1]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GGTCTC"
## 
## Slot "suffix":
## [1] "A"
## 
## Slot "vector":
## [1] ""
## 
## Slot "overhang":
## [1] "TCTT"
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "GGTGCGGTGGTTGCCTTTGGTAACA"
## 
## Slot "temperature":
## [1] 60.1791
## 
## Slot "difference":
## [1] 0.1790953
## 
## 
## [[2]][[2]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GGTCTC"
## 
## Slot "suffix":
## [1] "T"
## 
## Slot "vector":
## [1] ""
## 
## Slot "overhang":
## [1] "GTGA"
## 
## Slot "extra":
## [1] "CGAHN"
## 
## Slot "binding_sequence":
## [1] "AGAAAGAATGTGGATCAACACATCGAACTG"
## 
## Slot "temperature":
## [1] 60.11394
## 
## Slot "difference":
## [1] 0.06515983
## 
## 
## 
## [[3]]
## [[3]][[1]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GGTCTC"
## 
## Slot "suffix":
## [1] "A"
## 
## Slot "vector":
## [1] ""
## 
## Slot "overhang":
## [1] "TCAC"
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "GACGTAAACTTCTCTGTCGCCCAGG"
## 
## Slot "temperature":
## [1] 60.33165
## 
## Slot "difference":
## [1] 0.3316526
## 
## 
## [[3]][[2]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GGTCTC"
## 
## Slot "suffix":
## [1] "T"
## 
## Slot "vector":
## [1] ""
## 
## Slot "overhang":
## [1] "CGTC"
## 
## Slot "extra":
## [1] "TCAHN"
## 
## Slot "binding_sequence":
## [1] "ACCCGCCGGGTTTTCCGTACCG"
## 
## Slot "temperature":
## [1] 60.85613
## 
## Slot "difference":
## [1] 0.5244756
## 
## 
## 
## [[4]]
## [[4]][[1]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GGTCTC"
## 
## Slot "suffix":
## [1] "A"
## 
## Slot "vector":
## [1] ""
## 
## Slot "overhang":
## [1] "GACG"
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "CGTCGCGAAGTGGCGGTTATCA"
## 
## Slot "temperature":
## [1] 60.61078
## 
## Slot "difference":
## [1] 0.6107759
## 
## 
## [[4]][[2]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GGTCTC"
## 
## Slot "suffix":
## [1] "T"
## 
## Slot "vector":
## [1] ""
## 
## Slot "overhang":
## [1] "CTCT"
## 
## Slot "extra":
## [1] "TCAHN"
## 
## Slot "binding_sequence":
## [1] "GGCCTCTTTGGTGCGCCCGA"
## 
## Slot "temperature":
## [1] 60.47119
## 
## Slot "difference":
## [1] 0.1395836
## 
## 
## 
## [[5]]
## [[5]][[1]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GGTCTC"
## 
## Slot "suffix":
## [1] "A"
## 
## Slot "vector":
## [1] ""
## 
## Slot "overhang":
## [1] "AGAG"
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "ATCGACGGCGACGAACGTCC"
## 
## Slot "temperature":
## [1] 58.83656
## 
## Slot "difference":
## [1] 1.163437
## 
## 
## [[5]][[2]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GGTCTC"
## 
## Slot "suffix":
## [1] "T"
## 
## Slot "vector":
## [1] ""
## 
## Slot "overhang":
## [1] "ACGC"
## 
## Slot "extra":
## [1] "GCAHN"
## 
## Slot "binding_sequence":
## [1] "GTAGGCGCAGAAGTACAGACCGT"
## 
## Slot "temperature":
## [1] 59.13423
## 
## Slot "difference":
## [1] 0.2976635
## 
## 
## 
## [[6]]
## [[6]][[1]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GGTCTC"
## 
## Slot "suffix":
## [1] "A"
## 
## Slot "vector":
## [1] ""
## 
## Slot "overhang":
## [1] "GCGT"
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "CTGCATAACATTGAGCAGCAACTGC"
## 
## Slot "temperature":
## [1] 60.28916
## 
## Slot "difference":
## [1] 0.2891579
## 
## 
## [[6]][[2]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GGTCTC"
## 
## Slot "suffix":
## [1] "T"
## 
## Slot "vector":
## [1] "AAGC"
## 
## Slot "overhang":
## [1] ""
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "TTACAGCGCCATCAACTTGTCCAGC"
## 
## Slot "temperature":
## [1] 60.90327
## 
## Slot "difference":
## [1] 0.6141149
## 
## 
## 
## 
## Slot "newsequence":
## [1] "ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGGGAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACNDTCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCAGAAGAGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACGACCCAGTTCGATGTGTTGATCCACATTCTTTCTNDTCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTTGAAGAGCGTGACCTGAGCGGCTTTGTTGACGGTACGGAAAACCCGGCGGGTNDTGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCGGGCGGCAGCTATGTGTTTGTCCAGCGTTGGGAACACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGGTGATCGGGCGCACCAAAGAGGCCNDTGAAGAGATCGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTCGCCAGAGCCTGCCGTACGGCACTGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACNDTGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA"

The primers are generated for direct cloning into the Level 2 vector.
The function primer_add_level modifies the primers for individual cloning into Level 0 vectors and subsequent assembly in Level 2.
The parameters prefix, restriction_enzyme, suffix and vector can be set similar to the mutate-function.

primers_lvl0<-primer_add_level(primers,  prefix="TT", restriction_enzyme=recognition_site_bbsi, suffix="AA", vector=c("CTCA", "CTCG"))
primers_lvl0

## An object of class "Extended_Primerset"
## Slot "fragments":
## [[1]]
## An object of class "Fragment"
## Slot "start":
## [1] 2
## 
## Slot "stop":
## [1] 53
## 
## Slot "start_mutation":
## logical(0)
## 
## Slot "stop_mutation":
## [1] 51
## 
## 
## [[2]]
## An object of class "Fragment"
## Slot "start":
## [1] 54
## 
## Slot "stop":
## [1] 103
## 
## Slot "start_mutation":
## logical(0)
## 
## Slot "stop_mutation":
## [1] 101
## 
## 
## [[3]]
## An object of class "Fragment"
## Slot "start":
## [1] 104
## 
## Slot "stop":
## [1] 153
## 
## Slot "start_mutation":
## logical(0)
## 
## Slot "stop_mutation":
## [1] 151
## 
## 
## [[4]]
## An object of class "Fragment"
## Slot "start":
## [1] 154
## 
## Slot "stop":
## [1] 204
## 
## Slot "start_mutation":
## logical(0)
## 
## Slot "stop_mutation":
## [1] 202
## 
## 
## [[5]]
## An object of class "Fragment"
## Slot "start":
## [1] 205
## 
## Slot "stop":
## [1] 254
## 
## Slot "start_mutation":
## logical(0)
## 
## Slot "stop_mutation":
## [1] 252
## 
## 
## [[6]]
## An object of class "Fragment"
## Slot "start":
## [1] 255
## 
## Slot "stop":
## [1] 300
## 
## Slot "start_mutation":
## logical(0)
## 
## Slot "stop_mutation":
## logical(0)
## 
## 
## 
## Slot "oldsequence":
## [1] "ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGGGAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACGCGCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCAGAAGAGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACGACCCAGTTCGATGTGTTGATCCACATTCTTTCTCTGCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTTGAAGAGCGTGACCTGAGCGGCTTTGTTGACGGTACGGAAAACCCGGCGGGTGAAGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCGGGCGGCAGCTATGTGTTTGTCCAGCGTTGGGAACACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGGTGATCGGGCGCACCAAAGAGGCCAACGAAGAGATCGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTCGCCAGAGCCTGCCGTACGGCACTGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACTGCGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA"
## 
## Slot "primers":
## [[1]]
## [[1]][[1]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GAAGAC"
## 
## Slot "suffix":
## [1] "AA"
## 
## Slot "vector":
## [1] "CTCA"
## 
## Slot "overhang":
## [1] "AATG"
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "TCTCAGGTTCAGAGTGGCATTTTGCC"
## 
## Slot "temperature":
## [1] 60.36446
## 
## Slot "difference":
## [1] 0.3644563
## 
## 
## [[1]][[2]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GAAGAC"
## 
## Slot "suffix":
## [1] "AA"
## 
## Slot "vector":
## [1] "CTCG"
## 
## Slot "overhang":
## [1] "AAGA"
## 
## Slot "extra":
## [1] "TGAHN"
## 
## Slot "binding_sequence":
## [1] "GTCCGGGAATTTCGCTTCAAAAGTTGC"
## 
## Slot "temperature":
## [1] 60.56425
## 
## Slot "difference":
## [1] 0.1997931
## 
## 
## 
## [[2]]
## [[2]][[1]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GAAGAC"
## 
## Slot "suffix":
## [1] "AA"
## 
## Slot "vector":
## [1] "CTCA"
## 
## Slot "overhang":
## [1] "TCTT"
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "GGTGCGGTGGTTGCCTTTGGTAACA"
## 
## Slot "temperature":
## [1] 60.1791
## 
## Slot "difference":
## [1] 0.1790953
## 
## 
## [[2]][[2]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GAAGAC"
## 
## Slot "suffix":
## [1] "AA"
## 
## Slot "vector":
## [1] "CTCG"
## 
## Slot "overhang":
## [1] "GTGA"
## 
## Slot "extra":
## [1] "CGAHN"
## 
## Slot "binding_sequence":
## [1] "AGAAAGAATGTGGATCAACACATCGAACTG"
## 
## Slot "temperature":
## [1] 60.11394
## 
## Slot "difference":
## [1] 0.06515983
## 
## 
## 
## [[3]]
## [[3]][[1]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GAAGAC"
## 
## Slot "suffix":
## [1] "AA"
## 
## Slot "vector":
## [1] "CTCA"
## 
## Slot "overhang":
## [1] "TCAC"
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "GACGTAAACTTCTCTGTCGCCCAGG"
## 
## Slot "temperature":
## [1] 60.33165
## 
## Slot "difference":
## [1] 0.3316526
## 
## 
## [[3]][[2]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GAAGAC"
## 
## Slot "suffix":
## [1] "AA"
## 
## Slot "vector":
## [1] "CTCG"
## 
## Slot "overhang":
## [1] "CGTC"
## 
## Slot "extra":
## [1] "TCAHN"
## 
## Slot "binding_sequence":
## [1] "ACCCGCCGGGTTTTCCGTACCG"
## 
## Slot "temperature":
## [1] 60.85613
## 
## Slot "difference":
## [1] 0.5244756
## 
## 
## 
## [[4]]
## [[4]][[1]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GAAGAC"
## 
## Slot "suffix":
## [1] "AA"
## 
## Slot "vector":
## [1] "CTCA"
## 
## Slot "overhang":
## [1] "GACG"
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "CGTCGCGAAGTGGCGGTTATCA"
## 
## Slot "temperature":
## [1] 60.61078
## 
## Slot "difference":
## [1] 0.6107759
## 
## 
## [[4]][[2]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GAAGAC"
## 
## Slot "suffix":
## [1] "AA"
## 
## Slot "vector":
## [1] "CTCG"
## 
## Slot "overhang":
## [1] "CTCT"
## 
## Slot "extra":
## [1] "TCAHN"
## 
## Slot "binding_sequence":
## [1] "GGCCTCTTTGGTGCGCCCGA"
## 
## Slot "temperature":
## [1] 60.47119
## 
## Slot "difference":
## [1] 0.1395836
## 
## 
## 
## [[5]]
## [[5]][[1]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GAAGAC"
## 
## Slot "suffix":
## [1] "AA"
## 
## Slot "vector":
## [1] "CTCA"
## 
## Slot "overhang":
## [1] "AGAG"
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "ATCGACGGCGACGAACGTCC"
## 
## Slot "temperature":
## [1] 58.83656
## 
## Slot "difference":
## [1] 1.163437
## 
## 
## [[5]][[2]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GAAGAC"
## 
## Slot "suffix":
## [1] "AA"
## 
## Slot "vector":
## [1] "CTCG"
## 
## Slot "overhang":
## [1] "ACGC"
## 
## Slot "extra":
## [1] "GCAHN"
## 
## Slot "binding_sequence":
## [1] "GTAGGCGCAGAAGTACAGACCGT"
## 
## Slot "temperature":
## [1] 59.13423
## 
## Slot "difference":
## [1] 0.2976635
## 
## 
## 
## [[6]]
## [[6]][[1]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GAAGAC"
## 
## Slot "suffix":
## [1] "AA"
## 
## Slot "vector":
## [1] "CTCA"
## 
## Slot "overhang":
## [1] "GCGT"
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "CTGCATAACATTGAGCAGCAACTGC"
## 
## Slot "temperature":
## [1] 60.28916
## 
## Slot "difference":
## [1] 0.2891579
## 
## 
## [[6]][[2]]
## An object of class "Primer_MSD"
## Slot "prefix":
## [1] "TT"
## 
## Slot "restriction_enzyme":
## [1] "GAAGAC"
## 
## Slot "suffix":
## [1] "AA"
## 
## Slot "vector":
## [1] "CTCG"
## 
## Slot "overhang":
## [1] "AAGC"
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "TTACAGCGCCATCAACTTGTCCAGC"
## 
## Slot "temperature":
## [1] 60.90327
## 
## Slot "difference":
## [1] 0.6141149
## 
## 
## 
## 
## Slot "newsequence":
## [1] "ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGGGAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACNDTCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCAGAAGAGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACGACCCAGTTCGATGTGTTGATCCACATTCTTTCTNDTCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTTGAAGAGCGTGACCTGAGCGGCTTTGTTGACGGTACGGAAAACCCGGCGGGTNDTGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCGGGCGGCAGCTATGTGTTTGTCCAGCGTTGGGAACACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGGTGATCGGGCGCACCAAAGAGGCCNDTGAAGAGATCGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTCGCCAGAGCCTGCCGTACGGCACTGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACNDTGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA"

Objects of the classes “Primer”, “Primer MSD” and “Primerset” can have a slim textual output by using the function print_primer.

print_primer(primers_lvl0)

## Fragment 1
## Start 2, Stop 53, Length 52
## Forward
## TTGAAGACAACTCAAATGTCTCAGGTTCAGAGTGGCATTTTGCC
## Temperature of binding site:  60.36446  °C 
## Temperature difference:  0.3644563  K 
## Reverse
## TTGAAGACAACTCGAAGATGAHNGTCCGGGAATTTCGCTTCAAAAGTTGC
## Temperature of binding site:  60.56425  °C 
## Temperature difference:  0.1997931  K 
## 
## Fragment 2
## Start 54, Stop 103, Length 50
## Forward
## TTGAAGACAACTCATCTTGGTGCGGTGGTTGCCTTTGGTAACA
## Temperature of binding site:  60.1791  °C 
## Temperature difference:  0.1790953  K 
## Reverse
## TTGAAGACAACTCGGTGACGAHNAGAAAGAATGTGGATCAACACATCGAACTG
## Temperature of binding site:  60.11394  °C 
## Temperature difference:  0.06515983  K 
## 
## Fragment 3
## Start 104, Stop 153, Length 50
## Forward
## TTGAAGACAACTCATCACGACGTAAACTTCTCTGTCGCCCAGG
## Temperature of binding site:  60.33165  °C 
## Temperature difference:  0.3316526  K 
## Reverse
## TTGAAGACAACTCGCGTCTCAHNACCCGCCGGGTTTTCCGTACCG
## Temperature of binding site:  60.85613  °C 
## Temperature difference:  0.5244756  K 
## 
## Fragment 4
## Start 154, Stop 204, Length 51
## Forward
## TTGAAGACAACTCAGACGCGTCGCGAAGTGGCGGTTATCA
## Temperature of binding site:  60.61078  °C 
## Temperature difference:  0.6107759  K 
## Reverse
## TTGAAGACAACTCGCTCTTCAHNGGCCTCTTTGGTGCGCCCGA
## Temperature of binding site:  60.47119  °C 
## Temperature difference:  0.1395836  K 
## 
## Fragment 5
## Start 205, Stop 254, Length 50
## Forward
## TTGAAGACAACTCAAGAGATCGACGGCGACGAACGTCC
## Temperature of binding site:  58.83656  °C 
## Temperature difference:  1.163437  K 
## Reverse
## TTGAAGACAACTCGACGCGCAHNGTAGGCGCAGAAGTACAGACCGT
## Temperature of binding site:  59.13423  °C 
## Temperature difference:  0.2976635  K 
## 
## Fragment 6
## Start 255, Stop 300, Length 46
## Forward
## TTGAAGACAACTCAGCGTCTGCATAACATTGAGCAGCAACTGC
## Temperature of binding site:  60.28916  °C 
## Temperature difference:  0.2891579  K 
## Reverse
## TTGAAGACAACTCGAAGCTTACAGCGCCATCAACTTGTCCAGC
## Temperature of binding site:  60.90327  °C 
## Temperature difference:  0.6141149  K 
## 
## Input Sequence:
##  ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGGGAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACGCGCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCAGAAGAGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACGACCCAGTTCGATGTGTTGATCCACATTCTTTCTCTGCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTTGAAGAGCGTGACCTGAGCGGCTTTGTTGACGGTACGGAAAACCCGGCGGGTGAAGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCGGGCGGCAGCTATGTGTTTGTCCAGCGTTGGGAACACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGGTGATCGGGCGCACCAAAGAGGCCAACGAAGAGATCGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTCGCCAGAGCCTGCCGTACGGCACTGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACTGCGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA 
## 
## Modified Sequence:
##  ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGGGAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACNDTCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCAGAAGAGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACGACCCAGTTCGATGTGTTGATCCACATTCTTTCTNDTCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTTGAAGAGCGTGACCTGAGCGGCTTTGTTGACGGTACGGAAAACCCGGCGGGTNDTGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCGGGCGGCAGCTATGTGTTTGTCCAGCGTTGGGAACACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGGTGATCGGGCGCACCAAAGAGGCCNDTGAAGAGATCGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTCGCCAGAGCCTGCCGTACGGCACTGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACNDTGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA

The textual output can be printed into a file.

sink("primers.txt", append=FALSE, split=FALSE)
print_primer(primers_lvl0)
sink()