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.