Interestingly—there are no Esp3I sites in CoV2 after ORF1ab—considering that the Esp3I recognition sequence is cut out in no see’ms technology, it’s absence indicate that this sequence may have been optimized to be cloned using this enzyme.
In addition, a BamHi site is found in the end of the S, a BspE1 site is found at the end of the ORF3a site. an Age1 is found at the end of the E gene, in the middle of the M. The Pro in the PRRA is cut by BsaXi, used in RFLP and detects the integrity of the first R in the RRAR.
the BamHi, BspE1 and Age1 sites are unique in the entire genome.
The Pro and the +2 out-of-frame is enforced by the BsaXI enzyme. if the first R is disrupted or mutated away, the BsaXI will no longer cut. The 2 FauI sites in the S gene are compatible with each other, generating unique overhangs.
So does the 2 FauI sites after the BamHI site. However, the genes after the BamHI contained mostly unique restriction sites, including one genome-unique XhoI site. This XhoI site and the Genome-unique BseYI site after it defines a region of N that is different between CoV2 and
RmYN02/SARS.
Indicate that the genes after the S have been likely optimized to be manipulated using traditional restriction cloning, rather than type IIS cloning, separated by the BamHI site.
The post-S type II sites are all genome-unique.
A StuI site is found at the very end of the N. In addition, the TRS for ORF10 is missing. No evidence that it got translated......
Also found that there is a BseRI site encompassing the Pro and first Arg in the PRRA insert. The 2 BseRI sites are compatible with each other, and targets the complete S2 proportion of the S gene(full ectodomain). sigmaaldrich.com/technical-docu… This enzyme is a “Fuzyme” that can cut out
designated stop codons and allow two genes to be appended to each other. As there is an upstream alternative start codon in only the S gene of CoV2 and “RaTG13” encoding a peptide MFLLTTKRT that returned “protein tag” on search, it could easily be a subcloning site for the S1
Protein.
This enzyme is used for PCR-RFLP analysis since 1997.
Which solved the mystery behind the Pro of the PRRA insert—why it was absolutely required. It was both a Multiple Cloning Site(MCS), a Furin cleavege site(FCS) and a RFLP stability marker with 2 enzymes available for use. It is also a subcloning site for the HR1 and S2 domain,
A Subcloning site for the isolated S1 domain, and a Fuzyme site for fusing different protein tags/sequences to the C terminus of the S1 protein with a split codon. It also happen to be a Superantigen motif, a NRP1 binding site and a Heparan Sulfate binding site.
Why HR1 subcloning utility? advances.sciencemag.org/content/5/4/ea… in Apr 2019 they created EK1, a “pan-coronavirus fusion inhibitor” that needed to be evaluated against viral escape by enhancing HR1-HR2 interaction to outcompete EK1 binding. The reason behind the identical HR1 and the massive
Diffference in the nt sequence. Evolutionarily impossible.
And possibly the “massively improved ability to mediate fusion”.
As it turned out that the HR1 is neither stable nor optimally purified in-vivo, the only explanation is the conversion of a sequence into vector format, and possibly serial passage. Image
In a cellular (human lung CaLu-3 cells, or Vero E6 with RFLP purification).
Host or in nonhuman primates.
nature.com/articles/s4142…
Notice that their coronavirus fusion inhibitor with an optimized activity against CoV2, without modification, is actually developed long before CoV2 begins. A specific cure being made before the outbreak. @WlKD6iuxiZqBvm7
The BsmBI/Esp3I sites on ZC45, RaTG13 and CoV2. CoV2 is the only one that contained the convenient spacing needed to make no seem's technology easy. the HgaI is found at the end of the last Esp3I, unique across this fragment. it defines the beginning of the S gene. ImageImageImageImage

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More from @flavinkins

23 Sep
Shredding a "paper" who uses multiple fraudulent "evidence" to "debunk" the idea that SARS-CoV-2 is not a natural zoonosis.
centerforhealthsecurity.org/our-work/pubs_…
This "paper" was made by K_G Anderson, the creater of the fraudulent O-linked glycan claim to defend the WIV.
Claim 1: "e. SARS-CoV-1 and Middle East respiratory syndrome (MERS) have furin cleavage sites in their Spike protein." There are no furin cleavage sites in SARS-CoV-1, and MERS-CoV is a DPP4-using merbecovirus that have entirely different tropism and dynamics than Sarbecoviruses. Image
sciencedirect.com/science/articl…

fairdomhub.org/publications/5…
an absence of furin cleavage sites were found in Sarbecoviruses, the only ACE2-using beta-CoVs.
Read 48 tweets
22 Sep
In 2018, experiments with a novel recombinant SARS coronavirus substituted the typical TRS transcription leader and body sequences with a novel sequence (UGGUCGC) in an attempt to further reduce recombination in animal models as a live vaccine candidate (nature.com/articles/s4200…).
This TRS leader sequence, supposedly novel, is found starting at nucleotide 1465 in SARS-CoV-2 and could result, if utilized, in a novel viral RNA transcript that deletes part of the nsp2 protein of the ORF 1ab polyprotein.
It is also found at nucleotide 1446 in RaTG13, one of the viruses found in the Yunnan cave in 2013, which has been proposed as a precursor to SARS-CoV-2. It is found in that area in no other coronavirus. RaTG13 is fraudulent. Can’t infect a bat. The SRA is garbage.
Read 6 tweets
20 Sep
tandfonline.com/doi/full/10.10…

nature.com/articles/s4142…

advances.sciencemag.org/content/5/4/ea…
How Odd this EK1 is! Specific, confirmed, highly potent fusion inhibitor against CoV2. Online since early 2019??! Why Shi mentioned it as early as in Jan 31? Why not other drugs? Vaccines? Anything else? ImageImageImage
tandfonline.com/doi/full/10.10…
This was also the ZC45/ZXC21 homology paper. They have 4991 high connection—why never mentioned RaTG13? @WlKD6iuxiZqBvm7 @HimalayaGlobal @G_Translators
Read 4 tweets
20 Sep
@GentleMutt @BidoliNicola @MonaRahalkar @BillyBostickson @angoffinet @TheSeeker268 zenodo.org/record/4008432…
The real issue is that the raw data itself does not support the existence of that assembly. The rest of the SRA is a complete mess. Everything that have a match to a bat matches the genomic DNA instead of the mRNA. There were no bacterial reads which is
@GentleMutt @BidoliNicola @MonaRahalkar @BillyBostickson @angoffinet @TheSeeker268 Impossible for a “fecal swab”—every fecal sample on NCBI, if you search “bat feces” on SRA, with the Bacterial reads reduced below 5% in total cellular organisms, lacked any (confirmed) reads that can be traced to an RNA virus. There are other reads, including nearly 60% of a
@GentleMutt @BidoliNicola @MonaRahalkar @BillyBostickson @angoffinet @TheSeeker268 Non-homopolymer repeat sequence, which have no known biological mean of origin. It preprints.org/manuscript/202… have only ever been observed in significant quantities in 2 other datasets—the first one is a completely destroyed dataset (from tissue samples) with one amplicon of a
Read 19 tweets
19 Sep
biorxiv.org/content/10.110…
CRISPR gene editing to chop off the first 9 exons of ACE2, so only the short isoform remained, that can’t bind to CoV2 any more? @ch_zimmer
This isoform is a Carboxypeptidase and have ACE2 activity. So it will still work. It is no longer a receptor.
And will no longer allow infection.
Read 15 tweets
19 Sep
preprints.org/manuscript/202…
And every other part of that “raw data” points to complete RNA degradation—and complete virus destruction. Go to NCBI and search “bat feces”. Everything that had <5% bacteria lacked any virus—traced and confirmable through BLAST.
telegra.ph/RaTG13-07-06
The only set of amplicons that shared a well with the others in the same direction, possessed unusually low query coverage and appear to be in-house synthesized.
In fact, nearly all the bat “RNA” were partial match to the refseq RNA—indicative of the source of match being genomic DNA, filtered using a refseq/InSDC system and then separately merged into the dataset. The “bat mRNA” matches were always partial matches. If a clone is present,
Read 6 tweets

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