Puc19

Multiple displacement amplification assay was performed using the plasmid pUC19 (NEB, USA) as substrate. In brief, the pUC19 plasmid (NEB, USA) was mixed with random hexamer primers (NEB, USA) and incubated at 95 °C for 3 min and cooled to room temperature. The formed primed pUC19 DNA was added to the reaction mixture (25 μL, pH 7.5) contained 100 nM IME199 DNAP or 10 U phi29 DNAP (NEB, USA), 50 mM Tris -HCl, 10 mM (NH₄)₂SO₄, 10 mM MgCl₂, 500 μM dNTPs, 4 mM DTT, and incubated at 30 °C for 2 h. Electrophoresis and analysis were performed as described above.
The fidelity of IME199 DNAP was measured as previously described [15 (link)] with some modifications. In brief, IME199 DNAP or phi29 DNAP was incubated with 1 ng pUC19 (NEB, USA) at 30 °C for 8 h according to the above system. Amplification products were digested with restriction endonuclease SalI-HF (NEB, USA), heat inactivated, ligated, transformed into XL-10 competent cells (Vazyme, China), and plated onto Luria–Bertani solid plates containing ampicillin (50 μg/mL), X-Gal (0.8 mg) and IPTG (0.8 mg). Plates were incubated at 37 °C for 16 h and then the blue and white colonies were counted. In addition, the plasmids extracted from the white clones were sent to Ruibiotech (Beijing, China) for sequencing to identify the mutation site.

The SE LV is a self inactivated (SIN) LV expressing the enhanced green fluorescence protein (eGFP) gene under the control of an internal spleen focus-forming virus (SFFV) promoter. The different SARs elements (SAR1, SAR2, IgKIβ and Iβ) were inserted into the unique XhoI restriction site of the SE plasmid between the eGFP and the 3′LTR by standard cloning techniques to obtain the SE-SAR1Fw, SE-SAR1Rev, SE-SAR2Fw, SE-SAR2Rev, SE-IgKIβFw, SE-IgKIβRev, SE-IβFw and SE-IβRev. The different HS4-based LV and the IS2-LVs were constructed by first subcloning the LTR fragment into an XbaI site of pUC19 (New England Biolabs, Ipswich, MI, USA) to obtain pUC19-LTR. The different HS4 elements (HS4-Core, HS4-Ext and HS4-650) and the IS2 insulator were cloned into the unique Bbs1 site of the subcloned LTR to generate pUC19-modified-LTRs. The SE-CoreFw, SE-CoreRev, SE-ExtFw, SE-ExtRev, SE-650Fw, SE-650Rev, SE-IS2Fw and SE-ISRev LVs were constructed by removing the XbaI fragment from the SE vector backbone and replacing it with the different XbaI fragments from the pUC19- modified-LTR. All the constructs generated were verified by automated DNA sequence analysis and with multiple restriction enzyme digestions test.

Oxford Nanopore Sequencing was adapted from an established protocol^{8 (link)}. PE2 variants were extracted by direct PCR amplification from cultured yeast cells from a single 96 well with primers including the respective unique molecular identifiers (UMIs) (Supplementary Table 8). 25 cycles were performed prior to gel extraction and HiFi DNA Assembly into the pUC19 (New England Biolabs) which was previously digested (KpnI-HF, SpHI-HF). For each PCR amplification, approximately 1000 colonies were inoculated prior to plasmid purification. 100 ng of the plasmid pool was PCR amplified with 15 cycles NEBNext High Fidelity 2x PCR Master Mix and corresponding primers with binding regions outside of the UMIs and experiment specific barcodes. Thereafter, Oxford Nanopore sequencing was performed on the as previously described. Consensus reads were created by a previously described Python script^{8 (link)} and mutations were counted and identified with a custom Python script that will be deposited on GitHub prior to publication.