Domesticating the working vector
The library's working vector is pLVX-TetOne-Puro-GFP, which reads 6 BsaI and 2 BsmBI sites. This orders the same plasmid with all 8 taken out and confirms what arrives. It is a sitting of its own: the library run opens with the record it leaves behind.
- Parent
- pLVX-TetOne-Puro-GFP, Addgene 171123
- Plasmid
- 9,895 bp circular
- Sites taken out
- 6 BsaI, 2 BsmBI
- Bases changed
- 8
- Route
- the whole plasmid, ordered already changed
- Protein changed
- none
Have in hand
- pLVX-TetOne-Puro-GFP The parent plasmid, ordered from Addgene as deposit 171123 · circular, 9,895 bp
Leaves you with
- domesticated working vector The same plasmid with no BsaI and no BsmBI site, which the library run opens with · circular, 9,895 bp, no BsaI or BsmBI site
Keep in mind
Two sites sit at the same offset in the two long terminal repeats, and 610 bases around each one are identical between the copies. No primer, assembly overlap or Type IIS overhang is unique to one copy, so mutagenesis and assembly both fail at those two.
Leaving those two standing would make this a cheaper job. They are changed here because a published vector carries the change, and changing them is what puts the whole plasmid on the order form rather than a fragment of it.
3 of the sites sit in a coding sequence, where a synonymous codon takes the site away: PuroR and AmpR read exactly as the parent reads them.
The other 5 cost a stated base each. A published vector already carries the change made in both repeats, and another carries the change made at the BsaI site of the hPGK promoter. Neither group measured what the change cost, so neither number is stated here.
The BsmBI site in the hPGK promoter has no precedent anywhere. The base chosen neither makes nor breaks a CpG, which is the most this run can say for it.
Only BsaI and BsmBI were taken out. The result still reads BbsI 3, SapI 2, so an assembly whose own enzyme is one of those meets those sites unchanged.
- sitespass
- proteinpass
- other enzymeswarn
other enzymes warn: BbsI reads 3 sites, SapI reads 2 sites; neither was in this job
Materials
| Name | Supplier | Catalogue | Storage | Per run | Note |
|---|---|---|---|---|---|
| BsaI-HFv2 | New England Biolabs | R3733 | -20 °C | 10 units a digest | reads nothing in the result |
| BsmBI-v2 | New England Biolabs | R0739 | -20 °C | 10 units a digest | reads nothing in the result |
| BamHI-HF | New England Biolabs | R3136 | -20 °C | 10 units a digest | the control lane, one cut |
Equipment: Heat block or thermocycler holding 37 °C and 55 °C, Agarose gel rig and a transilluminator
Order the plasmid
Caution: A supplier whose clonal genes stop at 7 kb cannot build this plasmid; ask for a long-construct quote before ordering.
Expected result
- The supplier accepts the sequence and quotes a build.
Notes
- A direct repeat longer than 200 bases is a high-complexity sequence, which is priced and scheduled apart from the rest, so declaring it up front saves a rejected order.[1]
- The change at transcript position +4 goes into both repeats because a vector whose two repeats differ there changes what reverse transcription copies; the published vector made the same change in both.[2]
- The change at the hPGK promoter's BsaI site is the one a published vector already carries at the same base.[3]
- working-vector.gb is the depositor's own verified sequence with the changes above made on it.[4]
Confirm what arrives
Caution: Use a recombination-deficient strain and a 5 mL culture; a larger one is not worth the deletions it returns.
Expected result
- Colonies by the next morning at the latest, and a miniprep of a few micrograms.
Expected result
- The BsaI and BsmBI lanes look like the undigested lane.
- The control lane is one band at about 9,895 bp.
Notes
- An enzyme that did not cut and an enzyme nobody added look the same on a gel, which is what the control lane tells apart. The dose is 10 units an hour for one microgram.[5]
Expected result
- The consensus matches working-vector.gb at every base, both repeats included.
Holes
2 numbers this protocol would otherwise have to invent. None is filled with a guess, and each says below what it waits on: a bench, a shelf, a price, or a number nobody has published.
- H33 no sourced number — the change at transcript position +4 in both long terminal repeats: no titre for a lentiviral vector whose transcript start has been changed. Waiting on a bench to settle it. Filled by one packaging run titring this vector beside the parent it was built from.
- H34 no sourced number — the two changes in the promoter driving the transactivator: no expression figure for either changed base of the hPGK promoter. Waiting on a bench to settle it. Filled by a reporter assay reading this promoter beside the parent's.
Sources
- Twist Bioscience, gene synthesis sequence acceptance criteria · docs/research/working-vector-plvx-tetone.md https://www.twistbioscience.com/faq/gene-synthesis cited at sequence complexity
- Haellman, V. et al. (2021) Mammalian synthetic biology platform VAMSyB. Metabolic Engineering 66, 41-50 · docs/research/working-vector-plvx-tetone.md https://doi.org/10.1016/j.ymben.2021.04.003 cited at vector construction
- Peterman, N. et al. (2025) Nucleic Acids Research 53, gkaf528 · docs/research/working-vector-plvx-tetone.md https://doi.org/10.1093/nar/gkaf528 cited at Addgene 239691
- Addgene 171123, pLVX-TetOne-Puro-GFP · docs/research/working-vector-plvx-tetone.md https://www.addgene.org/171123/ cited at full sequence
- New England Biolabs, Protocol for a single restriction enzyme digest · docs/research/working-vector-plvx-tetone.md https://www.neb.com/en-us/protocols/0001/01/01/optimizing-restriction-enzyme-reactions cited at reaction setup