1. *Please provide feature table file in the right format (containing all the target features) obtained from DNannotator:

2. *Please give the gDNA sequence data file in FASTA format:

3. Input data description: (optional) (Please use the short words)

4. *Add extra bases to the feature sequence fragments at both ends of an exon. If the exon is shorter than 40 bp, 40 bp additional flanking sequences will be added at both ends. (This parameter should always be greater than 200)

5. Primer3 parameters:

 1）PCR Primer parameters:

• Primer Size  Min: Opt: Max:
• Primer Tm  Min: Opt: Max:
• Primer GC % Min: Opt: Max:
• Max Tm Difference:
• Max Poly-X: bp
• Max Self Complementary:
• GC Clamp:
• PCR Amplicon Size Range: bp (By this default setting, amplicon size will be tried for 501-1000 firstly, then, 301-500, 1001-2000... till a good design is found. If the target region size is larger than the maximum amplicon size, the region will be divided into several amplicons.)
• Mispriming Library human rodent_and_simple rodent none

 2)  Sequencing Primer parameters:

• Sequencing Primer Size:  Min: Opt: Max:
• Sequencing Primer Tm:  Min: Opt: Max:
• Sequencing Primer GC %: Min: Opt: Max:
• Sequencing Primer Max Poly-X: bp
• Sequencing Primer Max Self Complementary:

6. Other parameters:

• Splicing Signal Region Size: bp
• Low Quality Sequencing Data at The First: bp
• PCR Primer Picking Region Size: bp
• Quality Sequencing Size: bp (If an amplicon was larger than this, internal sequencing primers will be designed to cover the amplicon)

7. Universal tags tailed to primer's 5'end: (optional)
    (With universal tags, the default PCR product size will be forced to 300-500 bp. No sequencing primers will be designed.)

     No forward universal tag
     M13 universal forward primer: 5'-GTAAAACGACGGCCAG-3'
     Your custom forward universal primer:

     No reverse universal tag
     M13 universal reverse primer: 5'-CAGGAAACAGCTATGAC-3'
     Your custom reverse universal  primer:

8. Choice of outputs:

• Exon and flanking sequence output file in FASTA format
• Original Primer3 output file
• Detailed data of PCR products
• Summarized primer list table

9. Your operating system: Windows Linux or Unix or Mac

10. Please specify the email address where the design results can be delivered:

Email address *

11. Choice of email: several emails with original result data several emails with compressed result data in gzip format one email with compressed result data in gzip format

Your email box accepts message with size limit of  1 MB 2 MB 4 MB 6 MB per message

If your input data file is from a Mac machine, please be sure to check this box