Primer3 Input -version 0.4.0- Apr 2026

The basic structure looks like this:

For advanced use cases, pair Primer3 v0.4.0 with scripts (Perl, Python, or R) to parse output and iterate over multiple sequences. The input format described here remains compatible with later versions (v2.x, v3.x), making it a timeless skill for bioinformaticians. primer3 input -version 0.4.0-

PRIMER_PICK_LEFT_INPUT=200 PRIMER_PICK_RIGHT_INPUT=400 PRIMER_PRODUCT_SIZE_RANGE=150-250 Version 0.4.0 respects standard thermodynamic calculations (nearest-neighbor). The basic structure looks like this: For advanced

PRIMER_SEQUENCE_ID=E.coli_16S_region SEQUENCE=GTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGG PRIMER_TASK=pick_detection_primers PRIMER_PICK_LEFT_INPUT=100 PRIMER_PICK_RIGHT_INPUT=350 PRIMER_PRODUCT_SIZE_RANGE=180-220 PRIMER_OPT_SIZE=20 PRIMER_MIN_SIZE=18 PRIMER_MAX_SIZE=25 PRIMER_OPT_TM=60.0 PRIMER_MIN_TM=58.0 PRIMER_MAX_TM=62.0 PRIMER_MAX_DIFF_TM=2.0 PRIMER_MIN_GC=40.0 PRIMER_MAX_GC=60.0 PRIMER_GC_CLAMP=1 PRIMER_MAX_POLY_X=4 PRIMER_MAX_HAIRPIN_TH=47.0 PRIMER_SELF_ANY_TH=45.0 PRIMER_SELF_END_TH=45.0 pair Primer3 v0.4.0 with scripts (Perl