Non-functional DNA sequences

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Sometimes when designing DNA parts (e.g. promoters, RBSs etc.) there is a need to include spacer DNA that has no function other than to separate two other DNA sequences. I've often been unsure how to go about designing such a spacer sequence. To increase the likelihood of designing an adequately non-functional piece of DNA, I thought it would be a good idea to have a checklist of all the types of sequences you should make sure are not in the spacer sequence. This list is unlikely to be comprehensive so please add anything else you can think of. In addition if anyone has any sequences that they know to be entirely non-functional they might post them here.

Sequences to avoid

Don't forget to consider both the spacer DNA and the flanking DNA lest you create an undesirable sequence overlapping your DNA and the flanking DNA sequences.

Operator sequences

Repressor/activator binding sites, σ-factor binding sites and promoters should all be avoided if possible.

Start codons

e.g ATG


There is a lot of variability in RBS sequences. The E.coli consensus RBS sequence can be found here The Shine-Dalgarno sequence is -
Even a part of this sequence can induce translation if there is a start codon nearby. See Chen et al.[1] for more details.

Restriction Sites

Depending on your construction strategy, you may want to avoid certain restriction sites in your spacer sequence. If you are using the BioBricks standard assembly method for cloning, you need to avoid these restriction sites - EcoRI, XbaI, SpeI and PstI.

Methylation Sites

Depending on your cellular chassis, you may need to avoid sequences that could be methylated. For example, GATC will be methylated in some E. coli strains.

Repetitive Sequences

Either self-repetitive or similar sequence to other non-coding sequences can be problematic for a variety of reasons such as recombination and difficulty to PCR.

GC ratio

It seems that maintaining a GC ratio close to the native organism's would be desirable.


Structured sequences

If the sequence will be transcribed into RNA, you will want to make sure there isn't any significant secondary structure that may interfere with translation of your RNA or its function, if applicable.

Translational stop sequence

<bbpart>BBa_B0042</bbpart>: This sequence provides a stop codon in all 6 frames.

R1    TTA
F2     TAG
R3        TTA
F3         TAG
R2            TTA
F1             TAG



  1. Chen H, Bjerknes M, Kumar R, and Jay E. Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. Nucleic Acids Res. 1994 Nov 25;22(23):4953-7. DOI:10.1093/nar/22.23.4953 | PubMed ID:7528374 | HubMed [Chen]