Primer Design

Primers can be designed directly against the sequences annotated in CnidoSite, or against a sequence you paste in. Design is performed by primer3 (version 2.6.1) running on this server — your sequence is not sent to any third-party website. For reference, the primers released with this database were designed with an optimal melting temperature of 60°C (58–62 °C), an optimal primer length of 20 bp (18–24 bp), and a GC content between 40% and 60%, with self-complementarity restricted to limit primer-dimer formation; that parameter set is the CnidoSite published protocol preset below.

Template taken from CnidoSite: Clavulariidae_g10730 (Clavulariidae_g10730), 41,944 bp.
Template

Two ways to supply a template: pick a species and a gene / transcript ID, or paste your own sequence. If the gene ID is found it is used; the pasted sequence is used otherwise. A template must be at least 30 bp.

Load example sequence
Primer parameters
Advanced parameters (any field left empty keeps the preset value)

A value that is not a number is ignored. The product size range is applied only when both of its fields are filled in and the maximum is at least the minimum.

Melting temperature
Primer length and GC content
Product and output
Self-complementarity and probe
Download TSV
Design results sorted by primer3 penalty, lowest first

Template 41,944 bp · parameter preset CnidoSite published protocol (Tm 60 [58-62] °C, 20 [18-24] bp, GC 40-60%) · 5 primer pairs returned.

Pair Left primer (5'→3') Position Len Tm (°C) GC (%) Right primer (5'→3') Position Len Tm (°C) GC (%) Product (bp) Penalty
1 GTGCCGTTTTCGCCCATATC 16998–17017 20 59.971 55.000 GTAGGGGCTGGAACTAGGGA 17263–17282 20 60.031 60.000 285 0.060
2 CCGTCAAAACAACACGGCAT 2562–2581 20 59.970 50.000 GGCAAAAATCGGCCCGATAC 2742–2761 20 59.970 55.000 200 0.060
3 GTCGAATTGGCCTCGAATGC 30365–30384 20 59.972 55.000 GCCTAGCTTTGTGCCCTGTA 30455–30474 20 60.035 55.000 110 0.064
4 CGAATTGGCCTCGAATGCAG 30367–30386 20 59.971 55.000 GCCTAGCTTTGTGCCCTGTA 30455–30474 20 60.035 55.000 108 0.064
5 TGCGCCATTGACAGTGTTTG 32906–32925 20 59.969 50.000 CGACATATCGTTCCCCTGGG 33080–33099 20 59.966 60.000 194 0.064
Primer map pair 1 (lowest penalty) · product 285 bp at template position 16,998–17,282 · the template track shows a 1,500 bp window of the 41,944 bp template — the blue segment on the top bar is where that window sits, and every scale is in template coordinates · the two tracks are drawn at different scales

Left primer — binds the template strand as listed (5′→3′)Right primer — its reverse complement binds hereProductWindow drawn in the template track

whole templatethe 1,500 bp drawn in the track below · template 16,390–17,889 of 41,944Template1,500 bp windowproduct 285 bp · template 16,998–17,282product 285 bpleft primer, template 16,998–17,017right primer anneals here, template 17,263–17,28216,39016,76517,14017,51417,889Amplicon285 bpleft primer GTGCCGTTTTCGCCCATATC · 5′→3′right primer GTAGGGGCTGGAACTAGGGA · its reverse complement binds the template strandinterior 245 bp16,99817,06917,14017,21117,282
Left primer
GTGCCGTTTTCGCCCATATC
template 16,998–17,017 · 20 bp · Tm 59.971 °C · GC 55.000 %
Right primer
GTAGGGGCTGGAACTAGGGA
template 17,263–17,282 · 20 bp · Tm 60.031 °C · GC 60.000 %

Amplicon sequence (5′→3′ on the template strand, 285 bp) — blue is the left primer, red is where the right primer anneals. Copy from here to order or to check a base by eye.

GTGCCGTTTTCGCCCATATCTTGCGACATATTGAGAGTTATAAAGCAAGGCATATCTCGCTAGAATCCTTGAACAACGAGGAATATTTTGGTGTATAGTTTGTTTTTTACATGTCTAGAACTCGAGTTATTAACTGAGAACAATATCTCAGTCGTTCGTGGGATATTGATCTTGTTACAAAATATCTGTGTATATTACCGTCAGCTCGAAATTTCCTGTATATACGCTATTTTGTGCCGATAAACACCATCGTTAATGCAAATTTTCCCTAGTTCCAGCCCCTAC

The right primer is listed as its own 5'→3' sequence; what anneals to the template strand shown here is its reverse complement, so the highlighted stretch is complementary to the listed sequence. That is expected.

How to read this table. Position is a 1-based interval on the template, and both primers use the same convention: it is the stretch the primer occupies on the template strand (for the left primer, starting at its 5’ end; for the right primer, the region it anneals to, i.e. where the reverse complement of the listed sequence sits on the template). The two coordinates in a row can therefore be subtracted directly, and the amplicon view above marks both of them so you can check them by eye. Penalty is primer3’s weighted sum of the deviations from your constraints — lower is better, and the pairs are returned in that order. Tm is computed by primer3 from the SantaLucia thermodynamic parameters at the salt and primer concentrations in effect. The primer sequences themselves are 5’→3′ and can be ordered as they stand; the right primer anneals to the template as its reverse complement.

Specificity has not been assessed. This page only designs primers; it does not check whether they also anneal elsewhere in the genome or transcriptome. Once you have candidates, confirm each primer sequence with this site’s BLAST. The Tm and GC settings above are the design targets, and the annealing temperature still has to be optimised at the bench.

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