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Right now, the only supported assembly method is Gibson Assembly. BsaI and/or BpiI restriction enzymes could be added on the outsides of primers so each fragment anneals with its neighboring fragment through digestion and ligation rather than homology arms.
Functionality:
addition of BsaI and BpiI to the ends of the primers
consider the added BsaI and BpiI sequence when designing/selecting for primers
add additional seq on the 5' end, beyond the BsaI and BpiI sites
data-driven selection of overhangs between sets of fragments, eg (Potapov, 2018)
avoid duplicate or cross-specific overhangs
select for best binding overhangs, avoid weak overhangs like TATA
avoid assemblies where a set of valid and desired overhangs is impossible to select
reduce the allowable overlap between fragments to just the 4bp of the neighboring overhang
How overlap between fragments is calculated and how the overlap is selected will need to be included and overwrite the current consideration about overlap lengths.
The text was updated successfully, but these errors were encountered:
Right now, the only supported assembly method is Gibson Assembly. BsaI and/or BpiI restriction enzymes could be added on the outsides of primers so each fragment anneals with its neighboring fragment through digestion and ligation rather than homology arms.
Functionality:
Changes:
Most of these changes will be made in the primer3.go file: https://github.com/Lattice-Automation/repp/blob/master/internal/repp/primer3.go
How overlap between fragments is calculated and how the overlap is selected will need to be included and overwrite the current consideration about overlap lengths.
The text was updated successfully, but these errors were encountered: