Table 2.

Strategies to avoid toxicity of full-length constructs.

Approaches	Advantages	Disadvantages	Ref.
Low-copy number plasmid	Cryptic promoters are maintained at low level of expression	Low plasmid yield Flavivirus genome are often unstable	[46,47,48,49]
Bacterial artificial chromosome (BAC)	Minimization of toxicity by a strictly controlled replication leading to only one plasmid per cell. Stable maintenance of large DNA fragments	Low plasmid yield Manipulation of big DNA constructs	[48,56]
Inactivation of cryptic E. coli promoters (CEP)	CPEs are inactivated	Introduction of punctual mutation can disrupt the viral RNA structure and viral fitness	[50,51]
Intron insertion	Expression of toxic regions is interrupted in bacteria	Introduction of external sequences in the viral genome	[42,57,58]
In vitro ligation	Non-required propagation of full-length cDNA in bacteria	Viral genome is maintained in multiple fragments in bacteria Low ligation efficiency Low virus recovery efficiency	[52,53]
Gibson assembly or Circular polymerase extension cloning (CPEC)	Non-required propagation of full-length cDNA in bacteria Rapid assembly in one step	Viral genome is maintained in multiple fragments in bacteria Low virus recovery efficiency Error rate of the reaction can produce undesired mutations	[54,55,59]