Cloning Strategies for Polycistronic Constructs

Cloning of all constructs was performed using the pGEM-T easy vector (Promega) as an intermediate. First, a 300 base pair (bp) DNA fragment containing desired restriction endonuclease sites and 2A-encoding sequences were synthesized (Genewiz) and the sequences were listed in Table S1. A 6 bp stuffer sequence was placed in between each pair of endonuclease sites for gene insertion. Then sequences between AatII and BstXI on pGEMT-T were replaced with the synthesized DNA fragment PTE2A or 3P2A to obtain the cloning intermediates pGEM-T-PTE2A and pGEMT-T-3P2A. Next, genes of interest were PCR-amplified and inserted into the cloning intermediates one by one. The templates used for PCR are: pMXs-MGT^{33 (link)} for M, G, T, pLenti-GFP (Cell Biolabs, LTV-400) for GFP, pCSCMV-tdTomato (Addgene) for Td, and piRFP670-N1 (Addgene, #45457) for iRFP670. For quad-cistronic constructs, the restriction sites used for each gene position were: first, BamHI and NheI, second, SpeI and HindIII, third, XhoI and BspEI, last, XbaI and SalI. All bi-cistronic constructs were cloned in the intermediate plasmid pGEM-T-PTE2A, and the restriction sites used depended on the 2A sites in between the first and the second gene. For M-T2A-GFP, BamHI and HindIII were used for the insertion of M, and XhoI and SalI were used for the insertion of GFP. For all other bi-cistronic constructs in which P2A immediately followed the first gene, BamHI and NheI were used for the first gene, and the restriction sites used for the second gene vary by 2A sites: SpeI and SalI for M-P2A-GFP, XhoI and SalI for M-tPT2A-GFP and GFP-tPT2A-M, and XbaI and SalI for M-tPTE2A-GFP. All tri-cistronic constructs were also cloned in the intermediate plasmid pGEM-T-PTE2A, and the restriction sites used were: BamHI and NheI for the first gene, SpeI and HindIII for the second gene, and XhoI and SalI for the third (last) gene. A 6 bp kozak sequence ACCGCC was added right before the ATG start codon of the first gene in every construct and the stop codon TAA was added at the end of the last gene in every construct. Finally, the constructs were excised from pGEM-T and inserted into the pMXs retroviral vector (Cell Biolabs) with BamHI and SalI. To construct the quad-cistronic TPE2A constructs, P2A and T2A were PCR amplified and swapped based on PTE2A constructs.