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Removed (Banned)Apr 17, 2023
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Apr 17, 2023·edited Apr 17, 2023Author

Who are you, and why are you on here posting the entire history of promoters for cDNA? This post is about the viability of sequencing tech in finding specific nucleic acids in the natural world. 30nt is 0.1% of the SARS CoV 2 genome, and the natural genome doesn't have SV40 promoters. This post is not about the exact methods for cDNA clone production, and that line you cherry picked out of my article, right after, says "Unless it's from a conserved sequence or gene". This post is about how viable 1000-30nt chunks are for identifying a single virus, it's NOT about reverse genetics systems. I'm betting promoter genes are rather short, which is why you have picked it out? Thinking "Oh wait, I know a short sequence commonly used in reverse genetics that I like to pin everything in the world on, let me post that!"

Also: That 30nt sequence is the front of SARS CoV 2. Did I post the promoter gene from a well known reverse genetics systems, and say "I bet you'll never find this?" No, I did not. Funnily enough, you WON'T even find the one you're talking about IN A NATURAL VIRUS GENOME, in any other virus except SV40, barring recombination WITH A VERY SIMILAR VIRUS. You have NO IDEA what you are talking about (or, you do, and are trying to purposely make us all look like fools)

Are you that dude running around trying to convince everyone in the world that SV40 is in every virus on the planet? You better not be the same guy dude, you're about to get chewed the fuck out. If you're not the same guy, your little "omg look they use promoter genes too" post you have constructed here, concerning cDNA, is especially moronic, when your own material, says specifically:

"As noted earlier not all viral genomes are infectious, complicating the development of full length cDNAs and the recovery of recombinant viruses"

Many viruses are infectious, and that includes SARS CoV 2. Promoters are likely used just to help get enough accurate material, by making sure the polymerase can bind to the correct part of the plasmid. Is there a POINT to this wall of copy pasted text that I have a big feeling, you don't fully understand?

I SWEAR TO GOD if you're that same fucking guy I already banned, we're gonna have fucking problems dude. Your little "Ohh no SV40 is in EVERYTHINNNNGG" dumbshit has already fucked with every layperson trying to understand Kevin Mckernan's work, and I SEE your ass over there, trying to goad people into thinking "SARS CoV 2 is actually all these other viruses". I think you're a federal agent at this point, i.e. you're a modified limited hangout operator, trying to package real information with as much BS as you can muster. I TOLD YOU there was no SV40 in SARS CoV 2. The SV40 promoters in the shots, are FROM THE PLASMIDS, AND THEY'RE JUST PROMOTERS, THEY DON'T CODE FOR VIRAL FRAGMENTS. WHO THE FUCK ARE YOU???

Here: for anyone who is confused by this fucking hack - learn what a promoter is. They're pretty much just the "primers" of DNA, i.e. where the polymerase binds, BEFORE it starts transcribing a region of interest. Promoters are not copied by polymerases in a properly functioning plasmid or DNA segment

https://www.youtube.com/watch?v=FsDzsMpOIdk

https://www.khanacademy.org/science/biology/gene-expression-central-dogma/transcription-of-dna-into-rna/a/stages-of-transcription

https://en.wikipedia.org/wiki/Promoter_(genetics)

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Apr 17, 2023·edited Apr 17, 2023Author

Here's a perfect example of how this guy works. He shows up, posts a wall of text about "SV40 promoters being used in plasmids" without understanding that promoters are where polymerases bind, and aren't copied. Then, he finds something else showing that certain DOUBLE STRANDED NEGATIVE SENSE RNA VIRUSES (SARS CoV 2 is single stranded positive sense) need plasmids transfected with them, because their dsRNA by itself lacks the ability to translate a protein the natural virus usually contains before infection I THINK (hard to sift through this pile of crap, which is all he ever posts). That's what this paragraph means I'm pretty sure, it means: "they're missing this protein they normally need to do the transcription step, so we have to code for that too by putting plasmids in there"

"Isolated dsRNA genomes from Group V negative sense RNA viruses are not infectious because the genome sequence cannot be translated directly into a functional replicase complex needed to transcribe the incoming genomic RNA. As Group V virions contain a replicase protein complex essential for transcription, genome infectivity requires that cells be co- transfected with plasmids that express the genomic RNA and plasmids expressing transcripts that encode the replicase protein complex are needed for genome infectivity."

He then takes all this to mean: "Omg there's SV40 in every virus" or "Omg there is SV40 in everything produced off a plasmid".

"Sv40 + Plasmid = SV40 is in everything, all these viruses, and SARS CoV 2 as well"

I have trouble believing he is this stupid. FUCK OFF FED

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Apr 17, 2023·edited Apr 17, 2023Author

Banned. Here is the original post, so people can still read it:

"Beau

Writes Beau’s Substack

7 hr ago

So, how on earth, are we going to confuse viruses for something else? Remember that 30nt chunk I posted from SARS 2? I challenge you to take it, and start hunting for it in any other virus. I'd bet you'd never find it,……………..

https://www.jcvi.org/sites/default/files/assets/projects/synthetic-genomics-options-for-governance/Baric-Synthetic-Viral-Genomics.pdf

In the late 1970’s, a simple observation altered the course of virology research globally. Using a small dsDNA virus genome as a model (the Group I polyomavirus SV40) researchers cloned the viral genome into a bacterial plasmid and propagated the viral genome in bacteria. Upon isolation of the plasmid DNA from bacteria, restriction enzymes were used to excise the dsDNA viral genome, re-ligate the genome in vitro into a circular dsDNA and rescue virus following transfection of the genome into susceptible cells (Figure 2, Part C)(28). (Many advances in biotechnology have been, and continue to be, dependent upon this restrict-isolate-ligate technique, or variations of it.) Shortly

BARIC: SYNTHETIC VIRAL GENOMICS 41

Synthetic Genomics: Risks and Benefits for Science and Society

thereafter, full length cDNAs of positive strand RNA genomes were isolated following reverse transcription, the cDNAs cloned and propagated in bacterial plasmids, and following introduction of full length DNA into eukaryotic cells, recombinant viruses were rescued from the transfected cultures, although very inefficiently. The major problems with this approach were the difficulty in generating the appropriate termini, accurate genome sequence, problems in nuclear transport of the full length RNA genome, and splicing of the viral genomic RNA. To rectify the efficiency problems, bacteriophage promoters (T7, SP6, T3) were introduced upstream of the cloned viral cDNAs, allowing in vitro transcription of full length RNA copies of the viral genome using the appropriate phage RNA polymerase, nucleotide triphosphates, and other constituents (Figure 2, Part D). The full length RNAs, near exact replicas of the viral genome, were highly infectious upon transfection of susceptible host cells (Figure 2, Part E)(2, 65, 66). The ability to clone full length copies of viral genomes allowed for ease of manipulation of the genome and the introduction of specific mutations. Recovered viruses contained the introduced mutations that were encoded within the full length cDNA clones, providing a ready means of performing detailed genetic analyses of virus replication and pathogenesis.

As noted earlier not all viral genomes are infectious, complicating the development of full length cDNAs and the recovery of recombinant viruses. Isolated dsRNA genomes from Group V negative sense RNA viruses are not infectious because the genome sequence cannot be translated directly into a functional replicase complex needed to transcribe the incoming genomic RNA. As Group V virions contain a replicase protein complex essential for transcription, genome infectivity requires that cells be co- transfected with plasmids that express the genomic RNA and plasmids expressing transcripts that encode the replicase protein complex are needed for genome infectivity (Figure 3a). For most group V viruses, both genome negative and positive sense RNA infectivity can be booted using this approach with most investigators expressing full length plus (coding) strands from the initial transcript. The plus strands are transcribed to full length negative strands, which are used to express the appropriate set of mRNA encoding the full component of positive and negative strand RNAs. Using this approach

BARIC: SYNTHETIC VIRAL GENOMICS 42

Synthetic Genomics: Risks and Benefits for Science and Society

Schnell et al. successfully recovered the first recombinant negative stranded RNA virus, rabies virus, from a cloned cDNA, ushering in an era of Group V virus reverse genetics (68, 82). These findings were rapidly extended to other linear negative stranded RNAs like paramyxoviruses and then to segmented negative strand RNA viruses like influenza and other myxoviruses, and then select bunyaviruses and arenaviruses (20). Reverse genetic strategies for group V viruses with segmented genomes are most complex as multiple plasmids expressing copies of each genome segment must be simultaneously delivered to a cell along with the support plasmids encoding the transcriptase complex."

Now, for my words: Stay the fuck off my page. You'd think you'd have gotten the point when I banned your other account without saying anything. You're probably a disinformation agent (most likely) or, you're so stupid you can't comprehend the multiple debunks people have provided to your thoughts (less likely, since you know where to find reference genomes, and understand some points about genetics few seem to grasp, yet you still purposely misconstrue your "findings" to fit some bogus ass narrative you want to push)

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