This is a notebook to replace the NEB Split Set tool as it seems to no longer be working as of the week of 20250818. This uses the NEB overhang fidelity values from https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0238592#sec010. This also made use of code from Sriram Pendyala's GGA library notebook.

This is currently only usable for cases where you have fixed terminal overhangs for cloning into another vector (these will be the termini of the sequence entered). I have also only added three emperical sets here (SapI, BsaI, BsmBI) and will add more in the future.

You should only have to change the following values in chunk [6]: 1: ranges = [(1250, 1700), (1200, 1700),(1200, 1700)] (each pair represents the desired size of each fragment, and can be manually fiddled to get better ligation fidelity or to avoid splitting functional elements accross two segments)

2: gene=Seq("ggagG...aCaatg".upper())
(This is the sequence being broken up, for the current use, the ends are the terminal breakpoints)

3: empirical=bsaI_empirical (This specifies which set to use (bsaI_emperical, bsmbI_emperical, sapI_emperical)

The book should output:

1. The individual fragments to order (this still needs the restriction sites themselves added to generate the overhangs, a future iteration will do this for you).
2. The overhangs used
3. The average ligation fidelity of all junctions

I reccomend that you doublecheck the overhangs with NEB's ligation fidelity tool (this tool still seems to be working).