Synthetic promoter designer

Assemble a transcription-factor binding-site array with recommended spacings, motif-poor linkers, optional minimal promoters (TATA or gene cores), and cloning flanks for Promega pGL3, pGL4, and pNL luciferase vectors. Processing stays in your browser.

Pair with TF binding scanner to re-check the product, or CloneIT2 to place the insert on a map.

Reporter vector & cloning sites

Target vector 5′ restriction site 3′ restriction site

Transcription factor elements

Add one or more binding elements and set how many copies of each. Order top→bottom is 5′→3′ in the array.

Use lookup-table spacing between sites

Fixed gap (bp) if table off

Minimal / core promoter

Include minimal promoter (3′ of TF array)

Spacing lookup (selected pair)

Motif A Motif B

How this tool works

Sites are sampled from IUPAC consensus motifs, ordered as listed, and separated by spacers whose lengths come from a curated homotypic/heterotypic table (e.g. NF-Y–SREBP ~16–20 bp). Spacers are random DNA rescanned against a blacklist of common cores (TATA, CCAAT, Sp1, AP-1, CRE, κB, …) to reduce accidental off-target sites.

Vectors with built-in promoters

Promega pNL3.1 / pNL3.2 and pGL4.23[luc2/minP] already include a minimal promoter. pGL3-Promoter and pGL4.13 carry an SV40 promoter. For those backbones the tool disables an extra minimal promoter so you only design the upstream TF array. Promoterless vectors such as pNL1.2[NlucP], pNL1.1, pGL4.10, and pGL3-Basic still let you append a TATA or gene-style core.

Unexpected blacklist hits

After assembly the construct is scanned for common cores (TATA, CCAAT, Sp1, AP-1, CRE, κB, E-box, …). Hits that fall entirely inside intentional TF sites are ignored; everything else is listed with risk level, IUPAC pattern, matched DNA, strand, and which region it sits in (spacer, minimal promoter, or restriction flank). High-risk hits in spacers are the main concern — click Design promoter again to re-roll spacer DNA. Low-risk patterns (HRE, initiator, DPE) are short and often chance matches.

Limitations

Spacing values are literature-informed starting points for reporters, not universal rules.
Blacklist scanning cannot guarantee absence of every possible TF site or cryptic initiator.
Always verify MCS compatibility with your exact Promega lot and re-scan the final oligo/gene.