The procedure for the construction of entry and expression clones using the Gateway® system has been well described previously 14 (link), 20 (link), 23 (link), 40 (link). Briefly, all purified PCR products and synthesized ORF sequences with attB1 and attB2 sites were cloned into a Gateway® entry vector (pDONR221) using BP Clonase (Life Technologies, Grand Island, NY). The clones were colony selected from agar plates and the ORF inserts were sequenced verified using our Automatic Clone Evaluation (ACE) software. For clones to be accepted in our viral gene plasmid collection, they must not contain truncations, frameshifts, or more than one amino acid change when compared to the reference sequence. In addition, any nucleotide change in the sequences of att-sites was also not accepted because it may cause failure of BP or LR reactions 20 (link), 21 (link).
To construct the collection of expression clones, sequence verified viral ORFs in entry vectors were transferred into mammalian cell-free expression vectors (pANT7_cGST, pJFT7_nHalo or pJFT7_cHalo) using LR Clonase (Life Technologies, Grand Island, NY). All solution transfers were executed by the Biomek FX automation workstation, and each cloning step was assigned a specific barcode which is tracked by the FLEXGene database throughout the cloning process.
To construct the collection of expression clones, sequence verified viral ORFs in entry vectors were transferred into mammalian cell-free expression vectors (pANT7_cGST, pJFT7_nHalo or pJFT7_cHalo) using LR Clonase (Life Technologies, Grand Island, NY). All solution transfers were executed by the Biomek FX automation workstation, and each cloning step was assigned a specific barcode which is tracked by the FLEXGene database throughout the cloning process.
