qRT-PCR Primer Design
Input a Gene ID, cDNA or CDS sequence to design qRT-PCR primer pairs and flag 3-prime seed risk against homologous or similar CDS records.
Maximum input length is 250,000 bp. Candidate primer positions are searched within the first 2,000 bp; submit the relevant cDNA/CDS segment to design primers in another region.
Real-data example
TkA01G000070 · TK1151 · CDS 1170 bp
The example uses the site’s TK1151 reference CDS; the gene card provides identity and reference details.
Homologous or similar transcripts were searched against CDS records from the same reference; risk flags use the primer's 3-prime seed.
Input Resolution
Record: TkA01G000070 · Reference: TK1151 · Length: 1354 bp
Similar CDS / homolog candidates
| Record | Identity | Length | E-value | Bitscore |
|---|---|---|---|---|
TkAUNG075120 | 100.00% | 1170 | 0.0 | 2111.0 |
Candidate Primer Pairs
Primer pairs: 50Product range: 80–200 bpTarget Tm: 60.0 °C
Homologous-CDS 3-prime seed screening is not genome-wide specificity validation. Tm uses an approximate sequence formula. Exon junctions, dimers and hairpins are not checked; validate candidates for the experimental system.
| # | Score | Risk | Product | Forward primer | Reverse primer | 3-prime seed risk |
|---|---|---|---|---|---|---|
| 1 | 40.5 | medium | 80 bp 12-91 |
TGCTCCATTAGCCCTTAAGTGC12 · Tm 60.5 · GC 50.0% |
GGTATATGCGAGTGACTCCAAC70 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 2 | 40.5 | medium | 80 bp 255-334 |
AGAAGGTGAGAGGATTTCACGG255 · Tm 60.5 · GC 50.0% |
GTTGTTCTGAAGTCATCTCGGC313 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 3 | 40.5 | medium | 80 bp 497-576 |
CCCAACAGCAAGGCTTGATAAC497 · Tm 60.5 · GC 50.0% |
ATTGTATGCTTCACCCTCCACC555 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 4 | 40.5 | medium | 81 bp 11-91 |
GTGCTCCATTAGCCCTTAAGTG11 · Tm 60.5 · GC 50.0% |
GGTATATGCGAGTGACTCCAAC70 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 5 | 40.5 | medium | 81 bp 63-143 |
CACATTAGTTGGAGTCACTCGC63 · Tm 60.5 · GC 50.0% |
TCTGATCTCACCACAAATCCCC122 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 6 | 40.5 | medium | 81 bp 76-156 |
GTCACTCGCATATACCCTCTTC76 · Tm 60.5 · GC 50.0% |
GCTTTCCATCCTCTCTGATCTC135 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 7 | 40.5 | medium | 81 bp 123-203 |
GGGATTTGTGGTGAGATCAGAG123 · Tm 60.5 · GC 50.0% |
CCTTCCACAATGGTTGTACGTC182 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 8 | 40.5 | medium | 81 bp 310-390 |
ATGGCCGAGATGACTTCAGAAC310 · Tm 60.5 · GC 50.0% |
CATCTCTGACTGTCTACTTGCC369 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 9 | 40.5 | medium | 81 bp 497-577 |
CCCAACAGCAAGGCTTGATAAC497 · Tm 60.5 · GC 50.0% |
CATTGTATGCTTCACCCTCCAC556 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 10 | 40.5 | medium | 82 bp 2-83 |
AAAGTGGGTGTGCTCCATTAGC2 · Tm 60.5 · GC 50.0% |
CGAGTGACTCCAACTAATGTGC62 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 11 | 40.5 | medium | 82 bp 42-123 |
TAATGGAGGGGTAGTTCCAAGC42 · Tm 60.5 · GC 50.0% |
CCCTTCACTTAACCTTTCCCTG102 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 12 | 40.5 | medium | 82 bp 62-143 |
GCACATTAGTTGGAGTCACTCG62 · Tm 60.5 · GC 50.0% |
TCTGATCTCACCACAAATCCCC122 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 13 | 40.5 | medium | 82 bp 63-144 |
CACATTAGTTGGAGTCACTCGC63 · Tm 60.5 · GC 50.0% |
CTCTGATCTCACCACAAATCCC123 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 14 | 40.5 | medium | 82 bp 310-391 |
ATGGCCGAGATGACTTCAGAAC310 · Tm 60.5 · GC 50.0% |
GCATCTCTGACTGTCTACTTGC370 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 15 | 40.5 | medium | 82 bp 458-539 |
GAGTCAGAGTCGTTGGTTTGAC458 · Tm 60.5 · GC 50.0% |
TTCTCGGTTGGGTTCCAGATAG518 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 16 | 40.5 | medium | 83 bp 2-84 |
AAAGTGGGTGTGCTCCATTAGC2 · Tm 60.5 · GC 50.0% |
GCGAGTGACTCCAACTAATGTG63 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 17 | 40.5 | medium | 83 bp 9-91 |
GTGTGCTCCATTAGCCCTTAAG9 · Tm 60.5 · GC 50.0% |
GGTATATGCGAGTGACTCCAAC70 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 18 | 40.5 | medium | 83 bp 41-123 |
GTAATGGAGGGGTAGTTCCAAG41 · Tm 60.5 · GC 50.0% |
CCCTTCACTTAACCTTTCCCTG102 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 19 | 40.5 | medium | 83 bp 62-144 |
GCACATTAGTTGGAGTCACTCG62 · Tm 60.5 · GC 50.0% |
CTCTGATCTCACCACAAATCCC123 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 20 | 40.5 | medium | 83 bp 71-153 |
TTGGAGTCACTCGCATATACCC71 · Tm 60.5 · GC 50.0% |
TTCCATCCTCTCTGATCTCACC132 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 21 | 40.5 | medium | 84 bp 12-95 |
TGCTCCATTAGCCCTTAAGTGC12 · Tm 60.5 · GC 50.0% |
AGAGGGTATATGCGAGTGACTC74 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 22 | 40.5 | medium | 84 bp 70-153 |
GTTGGAGTCACTCGCATATACC70 · Tm 60.5 · GC 50.0% |
TTCCATCCTCTCTGATCTCACC132 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 23 | 40.5 | medium | 84 bp 456-539 |
GAGAGTCAGAGTCGTTGGTTTG456 · Tm 60.5 · GC 50.0% |
TTCTCGGTTGGGTTCCAGATAG518 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 24 | 40.5 | medium | 85 bp 11-95 |
GTGCTCCATTAGCCCTTAAGTG11 · Tm 60.5 · GC 50.0% |
AGAGGGTATATGCGAGTGACTC74 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 25 | 40.5 | medium | 85 bp 492-576 |
CAATTCCCAACAGCAAGGCTTG492 · Tm 60.5 · GC 50.0% |
ATTGTATGCTTCACCCTCCACC555 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 26 | 40.5 | medium | 86 bp 12-97 |
TGCTCCATTAGCCCTTAAGTGC12 · Tm 60.5 · GC 50.0% |
GAAGAGGGTATATGCGAGTGAC76 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 27 | 40.5 | medium | 86 bp 38-123 |
AAGGTAATGGAGGGGTAGTTCC38 · Tm 60.5 · GC 50.0% |
CCCTTCACTTAACCTTTCCCTG102 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 28 | 40.5 | medium | 86 bp 71-156 |
TTGGAGTCACTCGCATATACCC71 · Tm 60.5 · GC 50.0% |
GCTTTCCATCCTCTCTGATCTC135 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 29 | 40.5 | medium | 86 bp 492-577 |
CAATTCCCAACAGCAAGGCTTG492 · Tm 60.5 · GC 50.0% |
CATTGTATGCTTCACCCTCCAC556 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 30 | 40.5 | medium | 87 bp 9-95 |
GTGTGCTCCATTAGCCCTTAAG9 · Tm 60.5 · GC 50.0% |
AGAGGGTATATGCGAGTGACTC74 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 31 | 40.5 | medium | 87 bp 11-97 |
GTGCTCCATTAGCCCTTAAGTG11 · Tm 60.5 · GC 50.0% |
GAAGAGGGTATATGCGAGTGAC76 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 32 | 40.5 | medium | 87 bp 70-156 |
GTTGGAGTCACTCGCATATACC70 · Tm 60.5 · GC 50.0% |
GCTTTCCATCCTCTCTGATCTC135 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 33 | 40.5 | medium | 89 bp 9-97 |
GTGTGCTCCATTAGCCCTTAAG9 · Tm 60.5 · GC 50.0% |
GAAGAGGGTATATGCGAGTGAC76 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 34 | 40.5 | medium | 89 bp 458-546 |
GAGTCAGAGTCGTTGGTTTGAC458 · Tm 60.5 · GC 50.0% |
TTGCTGTTTCTCGGTTGGGTTC525 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 35 | 40.5 | medium | 89 bp 488-576 |
AGACCAATTCCCAACAGCAAGG488 · Tm 60.5 · GC 50.0% |
ATTGTATGCTTCACCCTCCACC555 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 36 | 40.5 | medium | 90 bp 2-91 |
AAAGTGGGTGTGCTCCATTAGC2 · Tm 60.5 · GC 50.0% |
GGTATATGCGAGTGACTCCAAC70 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 37 | 40.5 | medium | 90 bp 487-576 |
CAGACCAATTCCCAACAGCAAG487 · Tm 60.5 · GC 50.0% |
ATTGTATGCTTCACCCTCCACC555 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 38 | 40.5 | medium | 90 bp 488-577 |
AGACCAATTCCCAACAGCAAGG488 · Tm 60.5 · GC 50.0% |
CATTGTATGCTTCACCCTCCAC556 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 39 | 40.5 | medium | 90 bp 556-645 |
GTGGAGGGTGAAGCATACAATG556 · Tm 60.5 · GC 50.0% |
GTTTGAAGATCCTCTTGCACGC624 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 40 | 40.5 | medium | 91 bp 63-153 |
CACATTAGTTGGAGTCACTCGC63 · Tm 60.5 · GC 50.0% |
TTCCATCCTCTCTGATCTCACC132 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 41 | 40.5 | medium | 91 bp 456-546 |
GAGAGTCAGAGTCGTTGGTTTG456 · Tm 60.5 · GC 50.0% |
TTGCTGTTTCTCGGTTGGGTTC525 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 42 | 40.5 | medium | 91 bp 487-577 |
CAGACCAATTCCCAACAGCAAG487 · Tm 60.5 · GC 50.0% |
CATTGTATGCTTCACCCTCCAC556 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 43 | 40.5 | medium | 91 bp 653-743 |
GCCCATTACCTCCATCTTCAAC653 · Tm 60.5 · GC 50.0% |
ACGGGAACTTCTCCCATGTTTG722 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 44 | 40.5 | medium | 92 bp 62-153 |
GCACATTAGTTGGAGTCACTCG62 · Tm 60.5 · GC 50.0% |
TTCCATCCTCTCTGATCTCACC132 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 45 | 40.5 | medium | 92 bp 297-388 |
ACCGGAAGTTATTATGGCCGAG297 · Tm 60.5 · GC 50.0% |
TCTCTGACTGTCTACTTGCCTC367 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 46 | 40.5 | medium | 92 bp 458-549 |
GAGTCAGAGTCGTTGGTTTGAC458 · Tm 60.5 · GC 50.0% |
TGATTGCTGTTTCTCGGTTGGG528 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 47 | 40.5 | medium | 93 bp 458-550 |
GAGTCAGAGTCGTTGGTTTGAC458 · Tm 60.5 · GC 50.0% |
CTGATTGCTGTTTCTCGGTTGG529 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 48 | 40.5 | medium | 93 bp 484-576 |
CAACAGACCAATTCCCAACAGC484 · Tm 60.5 · GC 50.0% |
ATTGTATGCTTCACCCTCCACC555 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 49 | 40.5 | medium | 93 bp 980-1072 |
GGGCAAAGGACCATTTGAATCG980 · Tm 60.5 · GC 50.0% |
AGTGTCTTGAGTTACCGCCATC1051 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |
| 50 | 40.5 | medium | 94 bp 2-95 |
AAAGTGGGTGTGCTCCATTAGC2 · Tm 60.5 · GC 50.0% |
AGAGGGTATATGCGAGTGACTC74 · Tm 60.5 · GC 50.0% |
F: medium TkAUNG075120 R: medium TkAUNG075120 |