1 TmPrime: http://prime.ibn.a-star.edu.sg/
Accepted annealing temperature: 50–70 °C
Design conditions: Oligos with Tm of 55 or 65 +/- 2 °C

2 DNAWorks: http://helixweb.nih.gov/dnaworks/
Accepted annealing temperature: 58–70 °C
Design conditions: Oligo size: 50 nt Random; Annealing temp: 65 +/- 1 °C; No gaps are allowed in assembled gene

3 GeneDesign: http://baderlab.bme.jhu.edu/gd/
Accepted annealing temperature: no information
Users can not specify the oligonucleotide concentration and PCR buffer conditions.
Software does not provide the information of melting temperature derivation of oligonucleotide set.
Design conditions: Target oligo length: 60 bp; Chunk overlap 20 bp
Oligonucleotide design may fail when the sequence of consecutive oligonucleotides collides.

4 Gene2oligo: http://berry.engin.umich.edu/gene2oligo/
Accepted annealing temperature: no information
Design condition: Oligo Tm priority with of Tm of 55 or 65 +/- 4 °C
Oligonucleotide design may fail to converge.

5 Assembly PCR Oligo Maker: http://startrek.ccs.yorku.ca/~pjohnson/AssemblyPCRoligomaker.html
Accepted assembly (annealing) temperature: 50–60 °C
Accepted oligonucleotide length: 40–70 nt
Design condition: Maximum oligonucleotide length: 70 nt
Software does not provide the information of melting temperature derivation of oligonucleotide set.