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Add Golden Gate support to assemblies #1

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jjti opened this issue Dec 3, 2019 · 0 comments
Open
8 tasks

Add Golden Gate support to assemblies #1

jjti opened this issue Dec 3, 2019 · 0 comments

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@jjti
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jjti commented Dec 3, 2019

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

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.

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