Transformation mixtures were plated onto SD−His plates (Takara Bio) and incubated at 30 °C for 48 h. The lithosphere is relatively cold and rigid compared with the underlying asthenosphere, and so tectonic plates move as solid bodies atop the asthenosphere. Cells were washed with PBS and inoculated with viruses serially diluted in cell-culture medium at 1:10 dilution. Cells were washed with PBS 1 h after inoculation, and overlaid with 2% methylcellulose mixed at 1:1 with 2× DMEM supplemented with 20% fetal bovine serum, 200 units ml−1 penicillin and 200 μg ml−1 streptomycin. Both types of medium were supplemented with 10% fetal bovine serum, 1× non-essential amino acids, 100 units ml−1 penicillin and 100 μg ml−1 streptomycin. BHK-SARS-N cells were grown using MEM supplemented with 5% fetal bovine serum, 1× non-essential amino acids, 100 units ml−1 penicillin, and 100 μg ml−1 streptomycin, 500 μg ml−1 G418 and 10 μg ml−1 puromycin. RNAs were generated from YAC DNAs by in vitro transcription and electroporated together with an mRNA encoding the SARS-CoV-2 N protein either into BHK-21 cells (12 clones) or BHK-SARS-N cells (cells expressing the SARS-CoV N protein) (6 clones).

Then, 1-10 μg of in vitro transcribed viral RNA was electroporated together with 2 μg of the N gene transcript into BHK-21 cells and/or BHK-21 cells expressing the corresponding coronavirus N protein. For all coronaviruses, Https://tiktok.Com the fragment encompassing the viral 5′ untranslated regions (UTR) contained the T7 RNA polymerase promoter sequence immediately upstream of the 5′ end of the genome, and the fragment encompassing the 3′ end of the genome contained a unique restriction site (Extended Data Table 1) downstream of the poly(A) tail. Additionally, a similar protocol was performed on a PCR product of the N gene from corresponding coronaviruses, producing a capped mRNA that encodes the N protein. Extracted DNA was used as template for screening by multiplex PCR using the QIAGEN Multiplex PCR kit (QIAGEN) according to the manufacturer’s instruction. These vectors were amplified by PCR using primers containing at least 45-bp overlaps to fragments encompassing the 5′ or 3′ ends of different viral genomes (Supplementary Table 1). Amplification was performed using KOD Hot Start DNA polymerase (Merck Millipore) according to the manufacturer’s instructions. DNA mixtures were prepared beforehand and contained 100-200 fmol of 3′ and 5′ open ends for all fragments.

The YAC containing viral cDNA was cleaved at the unique restriction site located downstream of the 3′ end poly(A) tail (Extended Data Table 1). In brief, 1-2 μg of phenol-chloroform-extracted and ethanol-precipitated restricted DNA was resolved in nuclease-free water and used for in vitro transcription using the T7 RiboMAX Large Scale RNA production system (Promega) with m7G(5′)ppp(5′)G cap provided as described previously2. Fragments were released from the vector using the restriction enzymes described in Extended Data Table 3. Restricted fragments were subsequently gel-purified using standard methods27. She attacks Larry Trainor, using a corrupted version of the Negative creature (whether this is a construct created by her black power ring, or a revival of her former powers, is not made clear). Black Widow found herself fighting against Ant-Man, https://www.Connect.Lv/valentinaof4 who was hesitant to hurt her, which she assured him not to stress about as he didn’t stand a chance against her. Parliament initially contended that the colonists had virtual representation, but the idea “found little support on either side of the Atlantic”. A single colony was grown in 20 ml of SD−His liquid medium, 1 ml aliquots were removed and expanded in fresh medium every 12 h.

Yeast cells were first grown in YPDA broth (Takara Bio), and transformed cells were plated on minimal synthetic defined (SD) agar without histidine (SD−His) (Takara Bio). In parallel, isolated YACs containing full-length synthetic fragments 5 and 7, as well as SARS-CoV-2 and SARS-CoV-2-GFP viral genomes, were successfully transformed into E. coli TransforMax Epi300 electrocompetent cells (Epicentre) (data not shown). ZIKA virus strain PRVABC-59 (GenBank: KX377337) was provided by M. Alves and was cultured in Vero cells. Electroporated cells were co-cultured with susceptible mouse 17Cl-1, Vero B4 and Vero E6 cells to rescue rMHV-GFP (17Cl-1), rMERS-CoV and Https://valentinaof4.com/ rMERS-CoV-GFP (Vero B4), and rSARS-CoV-2, rSARS-CoV-2-GFP and synSARS-CoV-2-GFP (VeroE6). E. coli Epi300 cells containing the different synthetic fragments of SARS-CoV-2 in pUC57 or pUC57mini were grown at 30 °C to decrease the risk of instability and/or toxicity. HCoV-HKU1 synthetic fragments 1-4 were provided individually cloned into pUC57 by GenScript (Extended Data Table 3). MERS-CoV-Riyadh-1734-2015 (GenBank: MN481979) fragments 1-8 were synthesized and cloned into pUC57 by GenScript (Extended Data Table 3), containing homologous regions to TAR vectors pVC604 and pCC1BAC-His3. The SARS-CoV-2 synthetic DNA fragments were delivered cloned into pUC57 or pUC57mini by GenScript (Supplementary Data 1, Extended Data Table 3). Fragments 1.1, 1.2, 1.3 and 12 contained homologous sequences to pCC1BAC-His3.