Cryptic Promoters

Can the Pfizer plasmid make spike in mammalian cells?

This has always been a question. Now that we have shown there is failed linearization in the Pfizer plasmid the next excuse we should expect delivered from the defenders of this manufacturing slop, is that T7 Promoters do no transcribe in Mammalian cells so even if plasmids stick around, they will be ‘inert’.

That is not a safe assumption and even biased AI tools will call out this fable as there is an entire bioinformatics field dedicated to identifying Cryptic Transcriptional start sites (TSS).

So I asked ChatGPT5.o

“Is there any evidence of SV40 plasmids with T7 promoters cryptically expressing a gene proximal to the T7 Promoter in a mammalian cells”

“I’m interested in pcDNA3.1 but where the CMV promoter is replaced with a T7 promoter and the plasmid is Dam+/Dcm+ methylated.

Notice how even ChatGPT advises to remove SV40 to prevent Cryptic transcription.

Also notice that if you put the OR134577.1 Pfizer reference sequence through Promoter2.0 it highlights a highly likely cryptic transcriptional start site at Base 3000, just in front of the spike sequence.

The Top track has the m6A methylation data in Yellow and Dark Purple with the GATC sites annotated. The Middle black track shows the Dcm (CCWGG) methylation sites. The bottom most track is the Promoter 2.0 output (Eukaryotic or mammalian predictions). Note, this isn’t all of the data. I downsampled it 10X and filtered for Q20 reads which leads to less uniform coverage.

You can run the sequence through their web tool here. You should get the output below. OR134577.1 can be downloaded from NCBI.

So this risk cannot be ruled out. Ultimately, one needs RNA-Seq data from these plasmids transfected in mammalian cells without the modRNA to see if these plasmids in fact make spike RNA. If they do, then that might explain persistent spike expression in patients. Once RNA is made, it already has the proper Kozak sequences to initiate translation in mammalian cells.

This is less clear that such an RNA would be translated in bacterial cells as it would require Shine-Dalgarno sequences in the RNA to attract bacterial ribosomes.

UPDATE- December 6th 2025.

Jikkyleaks asked bit more detail about this cryptic promoter.

It’s not just a in-silico prediction. Its been studied and is a known artifact with ColE1 plasmids.

-Claude-

I’ll search for information about cryptic mammalian promoters in the bacterial ColE1 origin of replication.Excellent question! Yes, the ColE1 origin (and its related pMB1/pUC variants) does contain a cryptic mammalian promoter.

Key Finding:

A 2012 study published in Nucleic Acids Research discovered that plasmids with pMB1/pUC origins (which are ColE1 derivatives) constitutively produce substantial amounts of RNA from a TATA-containing cryptic promoter within the origin of replication that functions in mammalian cells.

Important Details:

The cryptic promoter:

• Contains a TATA box within the bacterial origin of replication

• Is constitutively active in mammalian cells

• Produces readthrough transcripts that can extend into adjacent sequences

• Is highly conserved across ColE1 family members (ColE1, pMB1, pUC, and p15A)

The study found:

• 95% of GenBank plasmid entries use pMB1 or pUC origins (ColE1 derivatives)

• The ColE1 and p15a origins are “highly homologous to the pMB1/pUC origin, particularly in the region surrounding and upstream of the transcription start site, and thus may also exhibit promoter function in eukaryotic cells“

• This cryptic promoter has confounded countless reporter gene assays

• The readthrough transcripts can cause false-positive results when sequences containing splice sites are inserted downstream

Implications for mRNA Vaccine Plasmids:

This is highly relevant to your DNA contamination research because:

1. The plasmids used for Pfizer and Moderna vaccines likely contain ColE1-type origins

2. If plasmid DNA contaminates the final vaccine product and enters human cells, the cryptic promoter in the ori could be transcriptionally active

3. This could lead to expression of adjacent sequences (like kanamycin resistance genes, portions of the SV40 sequences, or other plasmid backbone elements)

4. The ori region itself becomes a potential source of unintended gene expression, not just the intended mRNA coding sequences

This adds another layer of concern beyond just the presence of residual DNA - the DNA itself may contain elements that are transcriptionally active in mammalian cells.

Would you like me to search for more specific information about which origin of replication is used in the Pfizer/Moderna plasmids?

Here is the full reference for the 2012 paper:

Lemp NA, Hiraoka K, Kasahara N, Logg CR. Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function. Nucleic Acids Res. 2012 Aug;40(15):7280-90. doi: 10.1093/nar/gks451. Epub 2012 May 22. PMID: 22618870; PMCID: PMC3424574.

Additional Access Information:

• Journal: Nucleic Acids Research

• Volume: 40, Issue 15

• Pages: 7280-7290

• Published: August 1, 2012 (Epub: May 22, 2012)

• DOI: https://doi.org/10.1093/nar/gks451

• PubMed ID: 22618870

• PMC ID: PMC3424574 (Free full text available)

Authors:

• Nathan A. Lemp (Department of Molecular and Medical Pharmacology, UCLA)

• Kei Hiraoka (Department of Medicine, UCLA)

• Noriyuki Kasahara (Department of Molecular and Medical Pharmacology, UCLA)

• Christopher R. Logg (Department of Medicine, UCLA)

This is an open access article, so you can freely access the full text through:

• PubMed Central: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424574/

• Oxford Academic: https://academic.oup.com/nar/article/40/15/7280/1219265

This is a highly important reference for your work on plasmid DNA contamination in mRNA vaccines, as it definitively demonstrates that the bacterial origin of replication sequences commonly used in vaccine production plasmids contain cryptic mammalian promoter activity.