Site-Directed Mutagenesis Primer Designer (Beta): Help

Design a defined DNA sequence carrying one or multiple nucleotide substitutions.

Enter a design

Paste or upload one original ACGT sequence, FASTA record or GenBank record. Use 80–100,000 bp of DNA. Files stay in your browser.
Choose the method matching your template and intended product.
For each change, enter Position (bp), Original base and New base. Use + Add change for another substitution and x to remove a row. Position 1 is the first uploaded base; all positions refer to this original orientation.
Set Maximum primer length and Target binding Tm if needed, then click Design mutagenesis primers. Review every change, primer and PCR reaction before ordering.

Choose the method

Overlap PCR produces edited linear DNA. Amplify the listed pieces from the original template, then fuse them using the reported outer primer pair. This does not require a dedicated mutagenesis kit. When all edits fit into the outer primer regions, a single PCR fragment may be sufficient. Making a plasmid from linear DNA requires a separate joining or cloning step.

Overlap assembly produces a circular design from separate PCR fragments. Use a compatible overlap-assembly protocol to join them. The complete original circular template is required, and the whole plasmid is included in the final sequence.

Inverse PCR uses a back-to-back primer pair to amplify the full plasmid as linear DNA. Circularize it and remove the starting template using a compatible protocol, such as the Q5 mutagenesis workflow. Nearby edits can share a primer; distant edits are assigned sequential rounds. Each round starts from the verified product of the preceding round. Do not combine the round-specific primer pairs in one PCR. These are not QuikChange overlapping-primer designs.

Example: two changes in one plasmid

For a 4,000 bp plasmid, enter the complete circular sequence and choose a circular method. Add rows for two desired positions, for example 250 and 1800, checking each Original base against that same input. These numbers are examples of coordinates, not a prescription for a particular mutation. The final sequence should still be 4,000 bp and contain both substitutions.

With Inverse PCR, read the number of rounds and the changes assigned to each round. When the report gives two rounds, use the first round’s verified product as the second round’s template. Keep coordinates referenced to the original input; do not renumber the second change after the first round.

How edits enter the primers

The designer keeps an exact 18–35 nt region at each primer’s 3′ end and incorporates the desired changes upstream. Nearby mutations are grouped so that separate binding regions do not collide. Overlap designs carry the rewritten region in the adjacent primers and retain it once in the product. Inverse-PCR primers have adjacent 5′ endpoints with no designed overlap.

The overlap setting is editable from 12 to 40 bp. A longer cluster of changes can increase the overlap; review the reported length and warnings. Each rewritten region is limited to 200 nt. Designs needing more than eight overlap-PCR fragments or insufficient unchanged binding sequence require a longer template or separate rounds. The default total-primer limit is 60 nt, editable from 40 to 120 nt. This is a design budget, not a universal biological cutoff. Inverse-PCR grouping considers the actual primer length, exact 3′ binding Tm and pairing; larger or poorly balanced groups are split into rounds. In overlap mode, very close edits must share a rewritten region; if it cannot fit, the tool asks for a larger primer limit or separate rounds.

Primer-length guidance

NEB’s Q5 guidance requires at least 10 template-matching bases at the 3′ end and recommends HPLC or PAGE purification for primers over 60 nt. Its multiple-site overlap guidance uses 18–20 nt shared overlaps as a starting point. Total primer length and overlap length are different quantities. This tool uses exact 18–35 nt 3′ binding regions and reports their Tm separately; full mismatched-primer thermodynamics are not calculated.

Coordinates and validation

The original base must match the supplied sequence. Duplicate positions, unchanged substitutions, out-of-range coordinates and ambiguous bases are rejected. Circular GenBank records cannot be silently treated as linear DNA. Substitutions preserve sequence length and coordinate numbering, including changes across circular base 1.

Read the results

All listed changes are combined in the final sequence. Review the primer table, reaction plan and any intermediate inverse-PCR rounds. Export primer TSV, edited FASTA, edited GenBank or the complete JSON report. GenBank exports label the changed bases but do not transfer imported annotations.

Tm is calculated for the exact 3′ matching region using the shared nearest-neighbor model, excluding rewritten or mismatched regions. It is not a full mismatched-primer Tm, a kit-specific prediction or a PCR annealing temperature. Follow the chosen protocol for annealing conditions and end joining.

If a design is rejected

For an original-base mismatch, check the 1-based coordinate, strand and exact sequence version. For a duplicate-position error, retain one row for that base. For insufficient binding sequence or a primer-length error, review the reported region and method before changing the length limit or separating changes. Increasing the limit does not remove synthesis or method constraints. Upload one record under 1 MB; a file containing multiple FASTA records is rejected.

Scope

This version supports 1–50 nucleotide substitutions in one defined target. Protein mutations, codon choice, insertions, deletions and combinatorial libraries are not implemented. Protein-level design will also need the coding DNA, reading frame and chosen codons; a protein sequence alone does not specify a unique DNA template. Hairpin/dimer thermodynamics and genome-wide specificity are not evaluated. Computational reconstruction does not establish experimental success.

Gibson Assembly Primer Designer (Beta) · Assembly PCR Primer Designer (Beta)

Use this result with other tools

Direct transfer: after a successful design, click Send to Primer Binding Checker. This opens Primer Binding Checker with the complete final construct, circular/linear topology, full forward/reverse primer sequences and their names. Added linkers and intended edits are part of that final sequence. If there are more than 15 pairs, choose a batch and send each batch separately.

In the checker: confirm the imported template and pair names, then click Check primer binding. Inspect the binding positions and predicted products for each pair. This maps primers on the final construct. To check the initial fragment PCRs, use each original template and its corresponding primer pair in a separate check.

For a multi-round inverse-PCR design, the final-construct transfer does not validate the template used in each intermediate round. Check each round against the template specified in the reaction plan.

Manual follow-up: copy the full primers into Primer Scan and choose the relevant host or custom reference to screen for other binding candidates. Export the final FASTA and open it in Restriction Site Analyzer to inspect cloning sites, or in ORF / Protein Translator to review coding sequence and reading frame. These destinations do not have a direct send button in this designer.

Send again if a transfer is older than 30 minutes or has already been used. Keep both pages in the same browser and site. If a new tab is blocked, allow this site’s pop-up or use the exported FASTA and primer TSV for manual entry.

Correcting highlighted input

When the main action rejects pasted sequence text, unsupported characters are highlighted in the field and the error message explains the problem. Correct the marked characters using the alphabet allowed by this tool, then run it again. The highlights update as you edit. A malformed FASTA or GenBank record may require a structural correction even when no individual character is marked.