In fusion cloning

The polycistronic anti-ChAT iRNA sequences were identified by the DSIR algorithm (Supplementary Fig. 1b). The same elimination steps discussed above were followed and the four most highly ranked sequences were selected and modified to mimic the endogenous mir17-19b [17 (link)]. Complete Scaffolds flanked by overlapping sequences of the pLenti-syn::hM4Di-CFP plasmid [3 (link)], including restriction site BsRGI and AscI flanking the 5′ and SalI flanking the 3′ end to offer the option of either restriction or In-Fusion cloning (Clontech). The DNA was synthesized and cloned into a pUC57 vector by Genewiz (https://www.genewiz.com). In-Fusion cloning (Clontech) was used to place scaffolds 3′ of the hM4Di-CFP open-reading frame of the pLenti-syn::hM4Di-CFP plasmid, digested with AscI and SalI. MirE1 and MirP constructs were also cloned into the pLenti-syn::mCherry plasmid in which the hM4Di-CFP open-reading frame was replaced by the mCherry open-reading frame via gene synthesis (Genwiz) and in fusion cloning. BrGI was used instead of AscI for digesting the plasmid before Infusion cloning (Clontech).

To construct the V583DEAD (dead/deah gene WT copy) and MTX^RV583DEAD (dead/deah mutated gene copy) complementation plasmid 1, primers 1 and 2 (tables S5 and S6) were designed with Xho I restriction sites. These primers flank the gene of
interest and were used to amplify the DNA sequence from the isolated genomic DNA of the WT and MTX^R strains.
In-Fusion cloning (TaKaRa Bio) was performed using primers 1 and 2 with at least 15–base pair complementary sequence for ligation into vector pGCP123 (49 (link)), which was also digested with
the same restriction enzyme. The pGCP123::V583DEAD and pGCP123::MTX^RV583DEAD plasmid was generated in E. coli DH5α, verified by sequencing, and transformed into E. faecalis as described previously (49 (link)). Similarly, the van operon without the two-component system vanRS from the VRE strain was cloned using In-Fusion cloning (Takara Bio). Plasmid 2 was used, and primers 3 and 4 (tables S5 and S6) were designed to incorporate the restriction sites Xho I and Bam HI. The genes EF_RS05155, EF_RS07835, and EF_RS09020 from both VRE and VRE MTX^R strains were also cloned using In-Fusion cloning (TaKaRa Bio) with plasmid 1 and primers 5 to 14 (tables S5 and S6). Inverse PCR was used to amplify the vector pGCP123, and vector overlapping regions were incorporated in the primers to amplify the genes instead of restriction sites.

Coding region of trwI/virB6 was amplified using PCR from pMAK3 plasmid (R388) and cloned into pBADM11 vector using In-Fusion cloning (Takara Bio). This clone was modified by addition of flexible linker composed of Gly-Ser-Gly and a Flag tag at the 3′ end of the trwI/virB6 gene by PCR amplification followed by In-Fusion cloning (Takara Bio). Mutations were introduced into the trwI/virB6-Flag by In-Fusion cloning (Takara Bio). R388ΔtrwI/virB6 was generated by incorporation of chloramphenicol cassette inside of trwI/virB6 gene by homologous recombination according to a protocol described previously^{41 (link),42 (link)} using the SW102 strain^{43 (link)}.
The mating assay was performed as previously described^{9 (link),44 (link)}. E. coli TOP10 strain containing R388ΔtrwI/virB6 plasmid and complementation plasmids expressing VirB6-Flag or mutants were used for mating assay as donor strains and E. coli DH5α as recipient strain. The conjugation frequencies were calculated as transconjugants per recipients. All experiments were performed three times. The data are expressed as mean ± s.d. For comparison of two groups, an unpaired t-test was employed as implemented at https://www.socscistatistics.com/tests/studentttest/default2.aspx. Unprocessed numbers are reported in the supplementary information.