The basic structure looks like this:
PRIMER_INTERNAL_OPT_SIZE=20 PRIMER_INTERNAL_MIN_SIZE=18 PRIMER_INTERNAL_MAX_SIZE=30 PRIMER_INTERNAL_OPT_TM=65.0 # Probe Tm should be 5-8°C higher than primers PRIMER_INTERNAL_MIN_TM=63.0 PRIMER_INTERNAL_MAX_TM=68.0 Here is a real-world input for amplifying a 200 bp region from a bacterial 16S rRNA gene: primer3 input -version 0.4.0-
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 primer3 input -version 0.4.0-
SEQUENCE=AGCTAGCTACGATCGATTCGATCGATCGATCGATCG Specify where primers can bind. Coordinates are 1-based. primer3 input -version 0.4.0-
PRIMER_MAX_MISPRIMING=12.0 PRIMER_MAX_END_MISPRIMING=6.0 PRIMER_NUM_RETURN=5 Running Primer3 v0.4.0 Save your input as input.txt . Then run: