[OP] All of our open expression plasmids have BbsI sites in the lacI sequence and should be domesticated for common Type IIS enzymes

[jcmolloy]

Describe the Problem

pTi and pOBL both have two BbsI sites in the LacI coding sequence and also BtgZI sites. pTi has additional BbsI sites on the backbone so is not compatible with Type IIS assembly schemes involving BbsI.

Screenshots/Images

Example from pOBL

Sequence Data

These are genbank files but .gb cannot be uploaded to Github issues, please change the extension when you download them.

ptir.txt
pti.txt
pobl1-t7_openvent.txt
ptier.txt

Expected State/Behaviour

All Reclone backbones to have no commonly used Type IIS recoginition sites.

SEVA removed the following from the WT backbone (then added some back so origin of replication is flanked by FseI and AscI; antibiotic marker by SwaI and PshA; and the cargo by PacI and SpeI): HindIII, PstI, XbaI, BamHI, SmaI, KpnI, SacI, SalI, EcoRI, SfiI, SphI, AvrII, PshAI, SwaI, AscI, FseI, PacI, SpeI, SanDI and NotI.

I assume that the following are safe to remove from pTi (or at least to try and check for deleterious effects):

BbsI
BsmBI
BtgZI
HindIII
KpnI
PshAI
PstI
SacI
SapI (except for the ones to make it uLoop compatible)
SpeI

Asked on Reclone Forum if the following are used for anything? I’m guessing not but they are directly in between parts, so just checking…
XbaI
AvrII

https://forum.reclone.org/t/pti-plasmid-backbone-domesticating-for-more-type-iis-enzymes/1373

Follow-Up Actions (Assignees, Labels)

• Design primers to modify all sites
• PCR all backbones to sequentially replace the recognition site
• Sequence verify
• Rename to differentiate from current versions
• Upload sequence data to github
• Distribute new version to all Hubs and Nodes, include in Addgene submission