An R package to ease primer design using Primer3.
You can install primex from github with:
# install.packages("devtools")
devtools::install_github("dieterich-lab/primex")library(primex)
exonSeqs <- c(
  exon1 = paste0(
    "CTCACCATGGATGATGATATCGCCGCGCTCGTCGTCGACAACGGCTCCGGCATGTGCAAG",
    "GCCGGCTTCGCGGGCGACGATGCCCCCCGGGCCGTCTTCCCCTCCATCGTGGC",
    "AGGCACCAG"
    ), 
  exon2 = paste0(
    "GGCGTGATGGTGGGCATGGGTCAGAAGGATTCCTATGTGGGCGACGAGGCCCAGAGCAAG",
    "AGAGGCATCCTCACCCTGAAGTACCCCATCGAGCACGGCATCGTCACCAACTGGGACGAC",
    "ATGGAGAAAATCTGGCACCACACCTTCTACAATGAGCTGCGTGTGGCTCCCGAGGAGCAC",
    "CCCGTGCTGCTGACCGAGGCCCCCCTGAACCCCAAGGCCAACCGCGAGAAGATGACCCAG"
  )
)
seqOpts <- seqSettings(seqId = "transcript2", seq = exonSeqs) 
p3Opts  <- p3Settings() %>%  
  primerTm(min = 58, optimal = 63, max = 67)
primers <- design(seqOpts, p3Opts)First two candidate pairs:
| first | second | |
|---|---|---|
| PRIMER_PAIR_PENALTY | 4.488355 | 4.494866 | 
| PRIMER_LEFT_PENALTY | 4.462257 | 0.032609 | 
| PRIMER_RIGHT_PENALTY | 0.026098 | 4.462257 | 
| PRIMER_LEFT_SEQUENCE | ACCAGGGCGTGATGGTGG | ATGGATGATGATATCGCCGC | 
| PRIMER_RIGHT_SEQUENCE | CATGTCGTCCCAGTTGGTGA | CCACCATCACGCCCTGGT | 
| PRIMER_LEFT | 117,18 | 6,20 | 
| PRIMER_RIGHT | 244,20 | 134,18 | 
| PRIMER_LEFT_TM | 65.462 | 63.033 | 
| PRIMER_RIGHT_TM | 63.026 | 65.462 | 
| PRIMER_LEFT_GC_PERCENT | 66.667 | 50.000 | 
| PRIMER_RIGHT_GC_PERCENT | 55.000 | 66.667 | 
| PRIMER_LEFT_SELF_ANY_TH | 6.11 | 0.00 | 
| PRIMER_RIGHT_SELF_ANY_TH | 0.00 | 0.00 | 
| PRIMER_LEFT_SELF_END_TH | 0.00 | 0.00 | 
| PRIMER_RIGHT_SELF_END_TH | 0.00 | 0.00 | 
| PRIMER_LEFT_HAIRPIN_TH | 43.01 | 27.69 | 
| PRIMER_RIGHT_HAIRPIN_TH | 32.56 | 43.06 | 
| PRIMER_LEFT_END_STABILITY | 9.4000 | 12.9000 | 
| PRIMER_RIGHT_END_STABILITY | 7.9000 | 7.9000 | 
| PRIMER_PAIR_COMPL_ANY_TH | 0.00 | 0.62 | 
| PRIMER_PAIR_COMPL_END_TH | 5.31 | 0.00 | 
| PRIMER_PAIR_PRODUCT_SIZE | 128 | 129 |