Pick Primer ThermoHybrid (Release 2011_05)
pick primers from a DNA sequence (by Ivano Zara )

Input data
Anealing temperature 60.0 Primer melting temperaure 64.0
[primer] = 0.350 microM [Na+] = 50.000 mM [Mg++] = 1.500 mM [dNTPs] = 0.800 mM
Sequence length is 1542 Central zone length is 2 (920 - 921)
Product size: Min = 100; Max = 1000
Optimal Size = 600 (20 Penality for 100 bp of difference from optimal size)
No repeat library specified
Not Analyse template hairpin-loop

You are trying our example 1) Input Only Main Sequence
In this example the main sequence is given by the ribosomal sequence R16 of E.Coli (length 1541 bp).
The regions where to build the primers are delimited with the symbols '[' and ']' in position 921.
The primers will be built, one on the left and another on the right of these symbols
Penalty graf
low ← dangerous → high
  0    10   20   30   40   50   60   70   80   90   100  110 ... 
 |----|----|----|----|----|----|----|----|----|----|----| ... 
n For
or
Rev
Primer Sequence (5' -- 3') Product
size bp
3'
Location
PrimerHetero
dimer
penalty
Product
penalty
Total
penalty
Termodinamics
and
penalty details
Structure
penalty
Bases
penalty
for size
1) For
 GGTGTAGCGGTGAAATGCG 
613 700 0 0.0 0.0 0.0 2.6 2.6
Rev
 TCATGGAGTCGAGTTGCAGA 
1313 0 0.1 0.0

2) For
 CAGGTGTAGCGGTGAAATGC 
613 699 0 0.0 0.0 0.0 2.6 2.6
Rev
 TCATGGAGTCGAGTTGCAGAC 
1312 0 0.1 0.0

3) For
 CAGGTGTAGCGGTGAAATGC 
614 699 0 0.0 0.0 0.0 2.8 2.8
Rev
 TCATGGAGTCGAGTTGCAGA 
1313 0 0.1 0.0

4) For
 TTCCAGGTGTAGCGGTGA 
618 694 0 0.0 0.0 0.0 3.6 3.6
Rev
 TCATGGAGTCGAGTTGCAGAC 
1312 0 0.1 0.0

5) For
 TCAACCTGGGAACTGCATCTG 
573 639 0 0.0 0.0 1.1 5.4 5.5
Rev
 TGTGTAGCCCTGGTCGTA 
1212 0 0.0 0.0

6) For
 TCAACCTGGGAACTGCATCTG 
572 639 0 0.0 0.0 1.1 5.6 5.7
Rev
 GTGTGTAGCCCTGGTCGTAA 
1211 0 0.0 0.0

Note: Before choosing the primer pair it is advisable to see in details ('details') the analysis of any penalties and structures.
It is also important to see the complete analysis of the amplicon (visualize the possible structures that prevent / slow down the hybridization of the primers or that prevent the polymerization)