Sequence (plasmid or PCR product)

Circular (plasmid) Linear (PCR product, fragment)

Insert sequence

Designs PCR primers that add restriction sites to both ends of the insert. Structure: extra bases + enzyme site + (optional Kozak) + insert-binding part. Choose enzymes present in the vector MCS and absent from the insert.

5′ enzyme 3′ enzyme Target Tm °C

Add Kozak (GCCACC) before ATG — for mammalian expression Keep the insert stop codon

Vector and insert

Gibson, In-Fusion and HiFi assembly add sequences identical to the vector ends (overlaps) to the insert ends so the enzyme mix joins them. Choose where to open the vector (a unique cutter) and overlap primers are designed for you.

Vector sequence (circular) Insert sequence

Enzyme to open the vector Overlap length Target Tm °C

Sequence

Minimum ORF aa

Cloning helper

Sequence (plasmid or PCR product)

Map

Virtual digest · agarose gel

Sites by enzyme

Insert sequence

Vector and insert

Sequence

Sequence (plasmid or PCR product)▶ Try an example

MapUnique cuttersCut once or twiceAll enzymes

Virtual digest · agarose gel

Sites by enzyme

Insert sequence▶ Try an example

Vector and insert▶ Try an example

Sequence▶ Try an example

Sequence (plasmid or PCR product)

Map

Insert sequence

Vector and insert

Sequence