Primer binding sites for deep sequencing as well as multiplex identifiers (MIDs) for sample bar coding were synthesized into the inner primer design to create fusion primers compatible with the 454 platform.
Poliovirus RNA genomes that contained deletions in the capsid-coding regions were synthesized in monkey kidney cells that constitutively expressed T7 RNA polymerase.
The coding sequences were synthesized using codons optimized genes for expression in Arabidopsis [ 69] by GenScript (GenScript, Piscataway, NJ, USA) (GenBank: KP967458-KP967462).
These coding sequences were synthesized and subcloned into pET-24a, bypassing the N-terminal T7-tag but using the C-terminal 6xHis-tag.
The GCL and TSR coding sequences were synthesized with flanking att sites and cloned in the destination binary vector pEarleyGate100 (pEG100) via gateway-assisted recombination [ 76], resulting in vector TG1 with a bar selectable marker gene [ 77, 78] (Fig. 2a).
In order to construct an expression vector containing the O-methyltransferase genes that were under the control of independent T7 promoters, the 1.13-kb and 1.12-kb DNA fragments, which contain the sbOMT1 and sbOMT3 coding regions, were synthesized and then cloned into the NdeI and HindIII sites on pET-22b, which resulted in pET22-sbCOM1 and pET22-sbCOM3, respectively.
Primers spanning the two coding exons of TIRAP were synthesized by Sigma genosys (Haverhill, UK).
To generate the TGF-β131 and TGF-β313 chimeric proteins, the coding sequences of both were synthesized and inserted into a T7-based expression vector.
The DNA sequences coding for the EGSs were synthesized by the polymerase chain reaction (PCR), using construct pTK112 DNA [ 19] as the template.
Genes coding for these enzymes were synthesized into the vector pUC57 (Genescript, NJ, USA) with codon usage optimized for expression in M. extorquens AM1 in which the codon usage frequency was calculated by counting codon frequency in a list of ORFs associated with the 129 highly expressed genes in both methanol-grown and succinate-grown M. extorquens AM1 cells.
