Gibson Cloning Primer Design Tool

The Gibson Assembly^® Primer Design Tool

PLEASE READ BEFORE BEGINNING!

The Gibson Assembly Primer® Tool has been developed to assist you in designing primers for the assembly of DNA fragments. Primers designed with the tool can be used for DNA assembly with the Gibson Assembly® HiFi kit and the Gibson Assembly® Ultra kit. To use the tool, you will input the vector and insert sequences and indicate how DNA fragments will be prepared. DNA fragments can be prepared using PCR amplification, restriction digestion, or synthesis. The tool designs primers that add 30 to 40 nucleotides of homologous sequence at the end of adjacent fragments. The homologous sequences allow for seamless DNA assembly using the Gibson Assembly® HiFi and Ultra kits.

The Gibson Assembly® Primer Design Tool can be used in the following browsers:

The Google Chrome™ browser
Firefox
Internet Explorer 10 (IE10)

For optimal results, use Google Chrome.

For more information about the Gibson Assembly® method and the Gibson Assembly® HiFi and Ultra Kits, visit codexdna.com

Start Now

I understand.

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Vector Sequence

To start the tool, please enter your vector sequence in FASTA format. Please note, the homologous overlap will be added by PCR. Therefore, if the vector or fragment IS NOT PCR amplified, the homologous sequence will be added to the adjacent fragment.

The primer length is default at 60 nt?
The homologous length sequence is default at 40 bp?

Enter Your Vector ?

Do you want to PCR amplify your vector?

Yes No

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2. Your Vector Input

How would you like to select the site to place insert(s)? ?

By Position By Restriction

By Position

By Restriction

• Click and drag the sequence to highlight the insertion location

• Please select a site or region for insertion

Do you want to regenerate your restriction site??

Yes No

How many restriction cut sites do you want??

1 2

Vector 3' End?

Restriction Site

• Invalid Restriction Site

Vector 5' End?

Restriction Site

• Invalid Restriction Site

Scan Sites

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4. Your Results

The graphic circular map mimics the final assembled construct. Vector is displayed in black and inserts are displayed in color. Sequence information provides complete assembled construct with vector sequence in lowercase and the insert sequences in uppercase with highlight color. Primers will be designed between each junction to create fragments for Gibson Assembly. Please read the warning as primer design is based on the sequence information. The success of assembly is subject to many considerations. Some sequence elements such as repetitive motifs, GC flux, or highly homologous sequences may interfere with assembly. Primers generated with these sequences may require additional design to increase the assembly efficiency.

Sequence Information (Vector sequence in lowercase, Insert sequence in UPPERCASE)

• Insert sequence 3 > 10kb.

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5. Your Summary

The final design of the assembled construct is displayed in cartoon map. Below are the primers provided by the Codex DNA Gibson Assembly Primer design tool. For your convenience, the primer information can be download as Excel file or PDF summary. Additional information regarding the Gibson Assembly® HiFi 1-Step Kit or the Gibson Assembly® Ultra Kit can be found in the Codex DNA website.

Primer Sequence (Annealing sequence in lowercase, Homologous sequence in UPPERCASE)

Download Primers:

•

• PDF Summary

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5. Your Summary

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