SfaAI (AsiSI) (10 U/µL)

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	The CDKN2A/p16-wild-type ORF was subsequently digested by the restriction enzymes XhoI and SgfI to construct the pTUNE-CDKN2A/p16-wild-type inducible vector. The positive clone was also sequenced for verification.

Protocol tips
The CDKN2A/p16-wild-type ORF was subsequently digested by the restriction enzymes XhoI and SgfI to construct the pTUNE-CDKN2A/p16-wild-type inducible vector. The positive clone was also sequenced for verification.

Publication protocol

HAdV4 (GenBank NO. KF006344.1) and HAdV7 (GenBank No. HQ659699), two clinical isolates, were kindly provided by Prof. Rong Zhou (Guangzhou Medical University). Recombinant HAdV4 and HAdV7 expressing SEAP were constructed according to previously described methods (Zheng et al., 2017). In brief, viral genomic DNA was extracted by sodium-dodecyl-sulfonate lysis (Sigma-Aldrich, St Louis, MO, United States) followed by phenol-chloroform extraction. The terminal regions of HAdV4 and HAdV7 genome, which were used as homology arms, were amplified by PCR and subcloned into pMD19T-Simple vectors (TaKaRa, Dalian, China) to obtain shuttle plasmids pT-Ad4(L+R) and pT-Ad7(L+R), respectively. A unique restriction site for BamHI was introduced between the two arms, and two restriction sites for AsiSI were introduced at the 5′ terminal of the left arm and the 3′ terminal of the right arm. After digestion with BamHI (Thermo Fisher Scientific, Waltham, MA, United States), linearized pT-Ad4(L+R) and pT-Ad7(L+R) were subjected to homologous recombination with viral genomes in E. coli BJ5183 competent cells (Agilent Technologies, Santa Clara, CA, United States) to obtain genomic plasmids pAd4 and pAd7, respectively. Subsequently, the homology arms for E3 deletion, which contain 500 base pairs (bp) upstream of HAdV4 E3 region, or 520 bp upstream of HAdV7 E3 region, or 710 bp downstream of HAdV4 E3 region, or 660 bp downstream of HAdV7 E3 region, were amplified by PCR. Two shuttle plasmids, p4E3LR and p7E3LR, were constructed by inserting the respective homology arms into pVAX1 plasmids (Thermo Fisher Scientific, Waltham, MA, United States). The ligated homology arms were released from p4E3LR and p7E3LR by digestion with BstZ17I and SgrAI (Thermo Fisher Scientific, Waltham, MA, United States). Meanwhile, pAd4 and pAd7 were linearized by digestion with BclI and EcoRI (Thermo Fisher Scientific, Waltham, MA, United States), respectively. The E3 regions were then deleted by homologous recombination between E3LR fragments and linearized pAd4 and pAd7 to obtain pAd4ΔE3 and pAd7ΔE3, respectively. To construct recombinant HAdV4 and HAdV7 reporter viruses, the coding sequences for SEAP flanked by a CMV promoter and a BGH poly(A) signal were amplified by PCR using pGA1-SEAP as the template (Zheng et al., 2017), and inserted into p4E3LR and p7E3LR to obtain shuttle reporter plasmids pGK43-SEAP and pGK73-SAEP, respectively. Finally, pGK43-SEAP and pGK73-SAEP were linearized by digestion with BstZ17I and SgrAI, whereas genomic plasmids pAd4ΔE3 or pAd7ΔE3 were linearized by digestion with SwaI (Thermo Fisher Scientific, Waltham, MA, United States), and pAd4-SEAP and pAd7-SEAP were constructed by homologous recombination between linearized shuttle reporter plasmids and genomic plasmids. To rescue HAdV4-SEAP and HAdV7-SEAP, pAd4-SEAP and pAd7-SEAP were linearized by digestion with AsiSI (Thermo Fisher Scientific, Waltham, MA, United States) and transfected into HEK293 cells (ATCC). After successfully rescued, HAdV4-SEAP and HAdV7-SEAP were propagated in HEK293 cells and purified using cesium chloride gradient centrifugation. The infectious virus titers were determined as described previously (Aste-Amezaga et al., 2004

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