The Primer3 package also provides two stand-alone command-line programs: oligotm calculates oligo melting temperatures and ntthal (nucleotide thermodynamic alignment) predicts the propensity of oligos to form dimers or hairpins.
A major limitation (especially in high GC rich templates such as in P. aeruginosa) to Gibson assembly is that the termini of the DNA sequence fragments to be assembled should not have stable single stranded DNA secondary structure, such as a hairpin or a stem loop, or repeated sequences, as this would directly compete with the required single-stranded annealing/priming of neighboring assembly fragments. So make sure to run your sequences in Scitools to look for such nuisance areas (Delta G for hairpins should be more than negative 10 and with a lower annealing temperature than that of primer:primer duplex. Comment by DonHK).
Fixing
Should also attempt to fix the hairpin (by synthesis or junction shifting)
jjti
commented
5 years ago
Primer3 for hairpins
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424584/
UNAFold for hairpins
http://unafold.rna.albany.edu/?q=DINAMelt/software
Note
http://miller-lab.net/MillerLab/protocols/molecular-biology-and-cloning/gibson-assembly/
Fixing
Should also attempt to fix the hairpin (by synthesis or junction shifting)