Use this when you have a DNA sequence and need forward and reverse PCR primers. You can use the basic primer settings, or add cloning overhangs and restriction sites when needed.
Useful Checks
The same validated sequence can also be opened in Restriction Site Analyzer, which helps connect primer design with cloning-site checks.
After generating primers, open the primer pair directly in Primer Binding Checker to check binding positions and expected amplicon size on the same template.
Use fixed primer length when you want simple primers of a set size, or use target Tm when matching primer melting temperature matters more.
In target Tm mode, forward and reverse primers can be different lengths because each binding region is adjusted separately toward the same Tm.
Restriction-site overhangs use built-in enzyme-specific flanking-base rules, while binding-region Tm stays focused on the bases that bind the template.
Minimum Inputs Needed
DNA sequence.
Primer length, unless the default primer length is suitable.
Optional Settings
Template topology
Use linear for normal DNA fragments. Use circular / plasmid for plasmids or when the target may cross the coordinate origin, meaning base 1 / the start of the entered sequence, not the biological ori.
Amplicon region (optional)
Enter the part of the template you want to amplify. Leave it blank to use the full sequence.
Target Tm, deg C (optional)
Use this if you want primers near a specific melting temperature. Leave blank if primer length matters more.
Forward / reverse custom 5' overhang
Add extra bases to the 5' end of a primer. These bases do not bind the template in the first PCR cycles.
5' flanking bases before restriction site
Extra bases before a restriction site can help digestion. Auto uses built-in enzyme-specific flanking-base rules and is suitable for most cloning designs.
Forward / reverse restriction site
Add an enzyme site to the primer. Search by enzyme name or type the site sequence.
Na+, Mg2+, primer, and dNTP settings
These change the Tm estimate. Keep defaults unless you know your PCR conditions.
How To Use
Paste or upload the DNA sequence.
Choose linear or circular topology.
Enter an amplicon region only if you want a specific part of the sequence.
Change primer length or target Tm only if needed.
Add overhangs or restriction sites only for cloning designs.
Click Design primers.
Understanding The Results
The main outputs are the forward and reverse primers, written 5' to 3'.
The binding-region Tm, GC%, length, and warning flags help you judge the design.
Overhangs and restriction sites are included in the primer sequence, but not in binding-region Tm.
If the primer has warnings, check it in Primer Binding Checker before ordering.
Accepted Input Formats
Raw DNA sequence.
FASTA. Header lines are ignored.
GenBank where supported. Bases are read from the GenBank ORIGIN sequence section; feature annotations, including ori/origin-of-replication annotations, are ignored.
.dna / SnapGene binary files are not supported. Export as FASTA or GenBank.
Assumptions And Limitations
This is a primer planning tool, not a PCR guarantee.
Tm is an estimate and depends on the salt and primer settings.
Long overhangs, restriction sites, and difficult templates may still need manual checking.
Always verify final primer sequences before ordering.
Example
Paste your insert sequence, keep the default primer length, add EcoRI to the forward primer and HindIII to the reverse primer if needed, then design and check the primers.
Use note: These tools are for research and educational planning. Check important calculations and sequence designs before ordering reagents or running experiments.