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← Primer database

Primer database

Chrnb4

cholinergic receptor nicotinic beta 4 subunit

Rattus norvegicus

PublicationShang et al. 2022

Sequences

Forward (5′→3′)

CCAGGACGGAGGACGGTGAAC

Reverse (5′→3′)

AGGATAGCCAGCGAGGTGATGAG

Amplicon

137 bp

estimated from mapping

Primer Tm (°C)

ConditionsForwardReverseAverageΔ F/R
qPCR master mix68.767.368.01.4
Primer-BLAST default64.763.864.20.9

qPCR master mix: Owczarzy 2004/2008 · 250 nM primer · 50 mM Na⁺ · 3 mM Mg²⁺ · 0.8 mM dNTP

Primer-BLAST default: SantaLucia 1998 salt corr. · 50 nM primer · 50 mM Na⁺ · 1.5 mM Mg²⁺ · 0.6 mM dNTP

Bold, underlined base = 3′ end (polymerase extension point).

Primer QC

GC% · Forward

66.7%

GC% · Reverse

56.5%

Self-dimer ΔG · Fwd (any position)

-0.2 kcal/mol

Self-dimer ΔG · Fwd (3′ end)

0.5 kcal/mol

Self-dimer ΔG · Rev (any position)

-0.3 kcal/mol

Self-dimer ΔG · Rev (3′ end)

No duplex found

Heterodimer ΔG (F×R, any position)

-0.2 kcal/mol

Heterodimer ΔG (F×R, 3′ end)

0.5 kcal/mol

ΔG at 37 °C · SantaLucia 1998 NN parameters · < −6 kcal/mol = significant dimer risk · "3′ end" is restricted to duplexes anchored at a primer's own 3′ terminal base (polymerase-extendable); "any position" is the most stable duplex anywhere in the primer. Estimated screening value. For comprehensive dimer analysis, validate with Primer3.

Source

PMID

36479306(Europe PMC)

Citation

Shang et al. 2022

Genomic mapping

Ensembl gene

ENSRNOG00000014427 ↗

NCBI Gene ID

25103

Assembly

GRCr8

Cross-check

✓ENSRNOT00000101543
✓ENSRNOT00000019458
✓ENSRNOT00000138086★
~251bpThis is the outer span across every isoform's exon here. They don't all agree on the exact boundary (alternative splice site), so hover a row's own box below for its real length.69bp40bp149bp45bp110bp991bp937bp
exon presentprimer binds hereconfirmed amplicongap = exon skipped~bp = boundary varies
How do I read this diagram?

Each row is one transcript, aligned on a shared axis. A gap means that isoform skips this exonic region. Each row's box is drawn at that isoform's own real exon length, which can be narrower than the column (alternative splice site); hover a box for its exact size. A ~amber header bp means isoforms don't all agree on this region's boundary, while a plain header means they do. Navy blocks mark where the primers bind, and green (with a ✓ by the name) means this isoform has a confirmed working amplicon, not just exon overlap. A row with a “‹ exons” / “exons ›” hint instead of any box means that isoform's exons exist but sit outside the current scroll position (a truncated isoform or alternate transcription start site) — click the hint to jump to them. Click a row to load it below.

★ = Ensembl canonical transcript (reference model, not necessarily the most expressed isoform)

Showing: ENSRNOT00000101543

40bp1411bp149bp27.2kb45bp3255bp110bp41.8kb991bp56.5kb937bp6FRPredicted from the amplicon's own GC%+length (von Ahsen et al. 2001, at the same qPCR buffer as the primer Tm above: 50 mM Na⁺ plus 3 mM Mg²⁺ less 0.8 mM dNTP, as a sodium equivalent), not the SantaLucia model used for primer Tm, which isn't valid at this length. A screening estimate for ranking amplicons — that relation was fitted on oligonucleotide duplexes, so applying it at amplicon length carries more error than its published ±1.9 °C, and it is not a melt-peak prediction.137 bp amplicon · predicted amplicon Tm 87.9 °C
Chrnb4 · ENSRNOT00000101543⚠ Single-exon · may amplify genomic DNA

Off-target specificity

Specific

No paired forward+reverse off-target amplicon found against nt (whole genome/transcriptome). Screen requires >=2 total mismatches including >=2 within the last 5bp at the 3' end to clear a hit (NCBI Primer-BLAST-style stringency); hits with >=6 total mismatches are dropped as implausible.

Efficiency measurements

Ideal range:Dilution series 1.90–2.10·LinRegPCR 1.70–1.90·unknown method: not scored
No measurements yet for this primer pair.

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