Using Primer Design Assistant

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Main Parameters
Input format	Single Sequence in fasta format or ASCII (Pure text) will be allowed. But the multiple sequences pasted or uploaded into PDA must be in fasta format.
Sequence(s) input or upload	The example (fasta format) file and the other one (text format) file are here.
Primer length:	Too long a primer length may mean that even high annealing temperatures are not enough to prevent mismatch pairing and non-specific priming. The appropriate length of primer we suggest is between 18 -25 bps.
PCR product size:	The size is between 150 - 600 bps. This kind of sequence length is suitable for sequencing and further applications.
Default Settings
Repeats	Any four continual nucleotides (AAAA, TTTT, CCCC, or GGGG) will be avoided for both forward and reversed primers. Continuous dinucleotide repeats, such as ‘ATATAT’, are also avoided.
C/G clamp	G or C on the end of 3' terminal
GC %	25% ~ 75%
Tm	Tm of forward and reversed primers restricted to be higher than 50°C
△Tm	restricted to be smaller than 5°C
Advanced Options
Dimer check:	This option turns on can avoid primer dimer formation.
Hairpin check:	This option turns on can avoid internal self- complementarity.
5' GC content check:	Check the GC% of 5' to add the ability to recognize the template and enhance the priming specificity.
3' GC content check:	Check the GC% of 3' to avoid mismatch to avoid mismatch.
Covered region:	By entering the start position and stop position, you can get the PCR product containing the segment you need.
What if no primers are found?
Solution:	Because this web application was designed to find the best pair sets for multiple sequences in same PCR condition, some sequences may not hit the best pairs. Please change the parameters of primer length or size of PCR product. Or you can turn off some advanced options, like primer terminal GC content check.