Multiple Site Saturation Mutagenesis Example 3

Chris Ulpinnis & Pascal Püllmann

2020-08-28

Multiple Site Saturation Mutagenesis – Active site

You can find the lastest version of this file at https://github.com/ipb-halle/GoldenMutagenesis/blob/master/vignettes/MSD3.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

Aspartic Acid - 137 - NDT
Aspartic Acid - 143 - NNN
Asparagine - 147 - DBK
Arginine - 232 - NDT
Serine - 234 - 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
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
#We will use a structure similar to the Point Mutagenesis workflow to insert different kinds of Multiple Site Saturations here.
mutations<-list(c(137,"NDT"), c(143,"NNN"), c( 147, "DBK"),c(232, "NDT"), c(234, "NDT"))
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] 141
## 
## Slot "start_mutation":
## logical(0)
## 
## Slot "stop_mutation":
## [1] 137
## 
## 
## [[2]]
## An object of class "Fragment"
## Slot "start":
## [1] 142
## 
## Slot "stop":
## [1] 236
## 
## Slot "start_mutation":
## [1] 143 147
## 
## Slot "stop_mutation":
## [1] 232 234
## 
## 
## [[3]]
## An object of class "Fragment"
## Slot "start":
## [1] 237
## 
## 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] "AAAG"
## 
## Slot "extra":
## [1] "CCGCTCAGAHN"
## 
## Slot "binding_sequence":
## [1] "ACGCTCTTCAACCCAACGGAAGC"
## 
## Slot "temperature":
## [1] 59.785
## 
## Slot "difference":
## [1] 0.5794564
## 
## 
## 
## [[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] "CTTT"
## 
## Slot "extra":
## [1] "GTTNNNGGTACGGAADBK"
## 
## Slot "binding_sequence":
## [1] "CCGGCGGGTGAAGAGACGC"
## 
## Slot "temperature":
## [1] 59.2169
## 
## Slot "difference":
## [1] 0.7831005
## 
## 
## [[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] "GGCA"
## 
## Slot "extra":
## [1] "GAHNCTGAHN"
## 
## Slot "binding_sequence":
## [1] "AACAATCTTCAGCCCTTTGCCATCTT"
## 
## Slot "temperature":
## [1] 58.81557
## 
## Slot "difference":
## [1] 0.401326
## 
## 
## 
## [[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] "TGCC"
## 
## Slot "extra":
## [1] "G"
## 
## Slot "binding_sequence":
## [1] "TACGGCACTGCCAGTGGCACT"
## 
## Slot "temperature":
## [1] 59.7062
## 
## Slot "difference":
## [1] 0.2937957
## 
## 
## [[3]][[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] "TTACAGCGCCATCAACTTGTCCAG"
## 
## Slot "temperature":
## [1] 58.868
## 
## Slot "difference":
## [1] 0.8382065
## 
## 
## 
## 
## Slot "newsequence":
## [1] "ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGGGAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACGCGCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCAGAAGAGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACGACCCAGTTCGATGTGTTGATCCACATTCTTTCTCTGCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTTGAAGAGCGTNDTCTGAGCGGCTTTGTTNNNGGTACGGAADBKCCGGCGGGTGAAGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCGGGCGGCAGCTATGTGTTTGTCCAGCGTTGGGAACACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGGTGATCGGGCGCACCAAAGAGGCCAACGAAGAGATCGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTNDTCAGNDTCTGCCGTACGGCACTGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACTGCGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA"

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] 141
## 
## Slot "start_mutation":
## logical(0)
## 
## Slot "stop_mutation":
## [1] 137
## 
## 
## [[2]]
## An object of class "Fragment"
## Slot "start":
## [1] 142
## 
## Slot "stop":
## [1] 236
## 
## Slot "start_mutation":
## [1] 143 147
## 
## Slot "stop_mutation":
## [1] 232 234
## 
## 
## [[3]]
## An object of class "Fragment"
## Slot "start":
## [1] 237
## 
## 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] "AAAG"
## 
## Slot "extra":
## [1] "CCGCTCAGAHN"
## 
## Slot "binding_sequence":
## [1] "ACGCTCTTCAACCCAACGGAAGC"
## 
## Slot "temperature":
## [1] 59.785
## 
## Slot "difference":
## [1] 0.5794564
## 
## 
## 
## [[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] "CTTT"
## 
## Slot "extra":
## [1] "GTTNNNGGTACGGAADBK"
## 
## Slot "binding_sequence":
## [1] "CCGGCGGGTGAAGAGACGC"
## 
## Slot "temperature":
## [1] 59.2169
## 
## Slot "difference":
## [1] 0.7831005
## 
## 
## [[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] "GGCA"
## 
## Slot "extra":
## [1] "GAHNCTGAHN"
## 
## Slot "binding_sequence":
## [1] "AACAATCTTCAGCCCTTTGCCATCTT"
## 
## Slot "temperature":
## [1] 58.81557
## 
## Slot "difference":
## [1] 0.401326
## 
## 
## 
## [[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] "TGCC"
## 
## Slot "extra":
## [1] "G"
## 
## Slot "binding_sequence":
## [1] "TACGGCACTGCCAGTGGCACT"
## 
## Slot "temperature":
## [1] 59.7062
## 
## Slot "difference":
## [1] 0.2937957
## 
## 
## [[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] "AAGC"
## 
## Slot "extra":
## [1] ""
## 
## Slot "binding_sequence":
## [1] "TTACAGCGCCATCAACTTGTCCAG"
## 
## Slot "temperature":
## [1] 58.868
## 
## Slot "difference":
## [1] 0.8382065
## 
## 
## 
## 
## Slot "newsequence":
## [1] "ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGGGAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACGCGCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCAGAAGAGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACGACCCAGTTCGATGTGTTGATCCACATTCTTTCTCTGCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTTGAAGAGCGTNDTCTGAGCGGCTTTGTTNNNGGTACGGAADBKCCGGCGGGTGAAGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCGGGCGGCAGCTATGTGTTTGTCCAGCGTTGGGAACACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGGTGATCGGGCGCACCAAAGAGGCCAACGAAGAGATCGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTNDTCAGNDTCTGCCGTACGGCACTGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACTGCGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA"

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 141, Length 140
## Forward
## TTGAAGACAACTCAAATGTCTCAGGTTCAGAGTGGCATTTTGCC
## Temperature of binding site:  60.36446  °C 
## Temperature difference:  0.3644563  K 
## Reverse
## TTGAAGACAACTCGAAAGCCGCTCAGAHNACGCTCTTCAACCCAACGGAAGC
## Temperature of binding site:  59.785  °C 
## Temperature difference:  0.5794564  K 
## 
## Fragment 2
## Start 142, Stop 236, Length 95
## Forward
## TTGAAGACAACTCACTTTGTTNNNGGTACGGAADBKCCGGCGGGTGAAGAGACGC
## Temperature of binding site:  59.2169  °C 
## Temperature difference:  0.7831005  K 
## Reverse
## TTGAAGACAACTCGGGCAGAHNCTGAHNAACAATCTTCAGCCCTTTGCCATCTT
## Temperature of binding site:  58.81557  °C 
## Temperature difference:  0.401326  K 
## 
## Fragment 3
## Start 237, Stop 300, Length 64
## Forward
## TTGAAGACAACTCATGCCGTACGGCACTGCCAGTGGCACT
## Temperature of binding site:  59.7062  °C 
## Temperature difference:  0.2937957  K 
## Reverse
## TTGAAGACAACTCGAAGCTTACAGCGCCATCAACTTGTCCAG
## Temperature of binding site:  58.868  °C 
## Temperature difference:  0.8382065  K 
## 
## Input Sequence:
##  ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGGGAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACGCGCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCAGAAGAGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACGACCCAGTTCGATGTGTTGATCCACATTCTTTCTCTGCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTTGAAGAGCGTGACCTGAGCGGCTTTGTTGACGGTACGGAAAACCCGGCGGGTGAAGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCGGGCGGCAGCTATGTGTTTGTCCAGCGTTGGGAACACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGGTGATCGGGCGCACCAAAGAGGCCAACGAAGAGATCGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTCGCCAGAGCCTGCCGTACGGCACTGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACTGCGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA 
## 
## Modified Sequence:
##  ATGTCTCAGGTTCAGAGTGGCATTTTGCCAGAACATTGCCGCGCGGCGATTTGGATCGAAGCCAACGTGAAAGGGGAAGTTGACGCCCTGCGTGCGGCCAGTAAAACATTTGCCGACAAACTGGCAACTTTTGAAGCGAAATTCCCGGACGCGCATCTTGGTGCGGTGGTTGCCTTTGGTAACAACACCTGGCGCGCTCTGAGCGGCGGCGTTGGGGCAGAAGAGCTGAAAGATTTTCCGGGCTACGGTAAAGGCCTTGCGCCGACGACCCAGTTCGATGTGTTGATCCACATTCTTTCTCTGCGTCACGACGTAAACTTCTCTGTCGCCCAGGCGGCGATGGAAGCCTTTGGTGACTGCATTGAAGTGAAAGAAGAGATCCACGGCTTCCGTTGGGTTGAAGAGCGTNDTCTGAGCGGCTTTGTTNNNGGTACGGAADBKCCGGCGGGTGAAGAGACGCGTCGCGAAGTGGCGGTTATCAAAGACGGCGTGGATGCGGGCGGCAGCTATGTGTTTGTCCAGCGTTGGGAACACAACCTGAAGCAGCTCAACCGGATGAGCGTTCACGATCAGGAGATGGTGATCGGGCGCACCAAAGAGGCCAACGAAGAGATCGACGGCGACGAACGTCCGGAAACCTCTCACCTCACCCGCGTTGATCTGAAAGAAGATGGCAAAGGGCTGAAGATTGTTNDTCAGNDTCTGCCGTACGGCACTGCCAGTGGCACTCACGGTCTGTACTTCTGCGCCTACTGCGCGCGTCTGCATAACATTGAGCAGCAACTGCTGAGCATGTTTGGCGATACCGATGGTAAGCGTGATGCGATGTTGCGTTTCACCAAACCGGTAACCGGCGGCTATTATTTCGCACCGTCGCTGGACAAGTTGATGGCGCTGTAA

The textual output can be printed into a file.

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