Since the size is a bit larger (9.7 kb) I have to clone it in fragments (subcloning). Overlap primers can be designed for PCR amplification.
9.6kb synthesis as such is not possible, please use superscript III RT enzyme because its longer transcript
i suggest you to clone PVY in two part and join them or try with overlapping PCR based method
you find in your PVY genome sequence if it is present bacterial promoter like sequence? if it is available your clone is not stable in Ecoli so you create silent mutation in promoter like sequences of PVY genome. after the clone was stable in Ecoli
sir i agree with you that but how can we design overlapping primers without knowing the sequence.? for overlapping primers we must have full length sequence then we will design 2-4 sets of primers for amplification with overlapping region so that we can ligate them in single large fragment.
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