Assembly PCR Primer Designer (Beta): Help
How to build assembly and overlap-extension PCR primers: select a kit, enter vector and fragment sequences, set overlaps and export results.
Start a design
- Choose a named assembly kit or Custom for a circular construct. Choose Overlap PCR for a linear fusion without a vector.
- Set the total fragment count. A vector counts as one fragment.
- For GeneArt or In-Fusion, choose the listed kit vector or Custom. Other assembly modes show your vector inputs directly. Paste raw DNA, or paste/upload one FASTA or GenBank record. Inputs stay in your browser.
- For a circular vector, enter the base after which PCR should open it. For example, 250 opens between bases 250 and 251. The final base opens before base 1. Leave blank only if the supplied sequence is already linear and blunt-ended.
- Enter insert sequences in the desired order, without added assembly tails. Use + Add fragment, the up/down arrows and x to adjust the list. Choose Reverse complement only for a fragment supplied in the opposite orientation. Check the named junctions, overlaps and linkers, then click Design assembly PCR primers.
Example: vector plus two or three inserts
For a 3,000 bp circular vector with 600 bp and 900 bp inserts, set Total fragments to 3. Put the 600 bp insert first and the 900 bp insert second. Enter 1500 in Linearize after base to place the insert chain between original vector bases 1500 and 1501. With no added linkers, expect a 4,500 bp circle. The reported sequence starts at the first retained vector base after the opening.
Review all three junctions: vector → insert 1, insert 1 → insert 2, and insert 2 → vector. For three inserts, set Total fragments to 4 and review the additional internal junction. Recheck kit guidance when the fragment count changes. Primer-added homology is merged at joining; do not add it again to the expected final size.
Overlaps and vector ends
Overlap defaults follow the selected kit and total fragment count. Custom and Overlap PCR start at an editable 20 bp. Each fragment has an editable overlap field beside its optional linker. Untouched fields follow the main overlap setting; individual edits are retained. Values outside kit guidance are flagged.
A verified linear reference uses its actual first and last bases as the ends. Base 1 in an arbitrary circular plasmid file does not establish an insertion site. A single sequence and position cannot describe staggered restriction ends; this simplified form accepts PCR opening or an already-linear blunt backbone.
Optional DNA linkers
Enter an optional linker beside its fragment to add DNA before the next fragment. Leave the linker blank for a direct join. Enter A, C, G and T in the final construct’s 5′ to 3′ direction (up to 200 bp); amino-acid linkers must first be converted to your chosen DNA sequence.
Between two PCR fragments, the full linker is included in the reverse primer of the left fragment and the forward primer of the right fragment, with the reverse primer using its reverse complement. Assembly leaves one copy. The linker counts toward the shared overlap; if it is longer than the overlap setting, the overlap increases to fit it and is reported in the result. At a prepared-vector junction, only the insert primer receives the linker, alongside homology to the unchanged vector end. Review long-oligo and kit-guidance warnings and the reading frame.
Kit vectors
GeneArt Seamless A13288 offers the verified 2,659 bp pUC19L reference (also sold as A13289). In-Fusion Snap offers its distinct 2,690 bp linear pUC19 control vector. Both use their recorded physical ends. NEBuilder HiFi uses a user-prepared backbone; its supplied pUC19 is a transformation control. The vector upload and sequence fields open automatically.
Primer and reaction settings
Target binding Tm is the desired melting temperature of the part that matches the PCR template; added 5′ tails are excluded. The default is 60 °C, editable from 40 to 75 °C. It is not the PCR annealing temperature. Expand Reaction settings to enter the reaction concentrations used for this estimate: Na+ equivalent, Mg2+ and total dNTP in mM, and each primer in nM. For example, 0.2 mM of each dNTP means 0.8 mM total.
Overlap PCR without a vector
Choose Overlap PCR, then enter at least two fragments in the order wanted in the linear product. Total fragments now counts only those pieces. There is no closing junction from the final fragment back to the first. The reaction plan lists the initial fragment primer pairs and identifies the first forward and last reverse primers for the final fusion. Keep the fragment PCRs separate from the final fusion step.
Read the result
Each primer contains a template-binding region and any added overlap or linker tail. Tm uses the binding region only. Prepare each PCR fragment separately; kit assembly joins them in the assembly reaction. Overlap PCR instead uses the first forward and last reverse primers for the final linear fusion. The exports contain the full assembled sequence and primer table.
Input sequences are kept as supplied, including matching terminal bases. Remove unwanted duplicate sequence before design. GenBank import extracts sequence only; exported features identify fragments and added linkers, not transferred genes.
Check sizes and primer pairs
Read each primer 5′ to 3′. Order the full sequence, including its tail, rather than just the binding region. A PCR-opened vector has its own primer pair; an already-linear prepared vector does not. Each insert has a pair. The listed PCR-fragment size can include added tails and can be longer than the original input fragment.
For a direct circular join, final size is retained vector length plus all supplied insert lengths plus one copy of each added linker. Check both end junctions as well as every insert-to-insert junction. Matching bases already present in two supplied fragments are retained as input sequence; the tool does not automatically trim pre-existing homology. Export primer TSV for the oligos, FASTA or GenBank for the construct, or JSON for the full report.
What to check
Follow all instructions for the selected kit, including fragment count, overlap, DNA preparation and reaction conditions. Review the exact vector variant and ends, reading frame and junctions. Computational design does not establish experimental success; hairpin/dimer thermodynamics and genome-wide specificity are not evaluated.
Gibson Assembly Primer Designer (Beta)
For nucleotide changes, use the Site-Directed Mutagenesis Primer Designer (Beta).
Input limits and common mistakes
Use one unambiguous A/C/G/T sequence per field, up to 100,000 bp each. Sequence uploads must be under 1 MB; the final assembly limit is 200,000 bp. Kit guidance can be more restrictive. Export SnapGene .dna files as FASTA or GenBank before importing.
A circular-record error with a blank opening position means the tool has been asked to treat a circular record as an already-linear vector. Enter the intended opening coordinate. If the result is larger than expected, check for duplicated source overlap or a linker entered in more than one place. If the insert is reversed, check its supplied strand and Orientation in the construct. Re-run the design after corrections.
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.
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.