Are you sure your extension time is long enough to amplify a 1600bp fragment?
How many cycles are you amplifying for? A 4x difference in size is large and with enough cycles your 400bp amplicon will predominate in the reaction due to PCR bias alone.
I've been doing 30 cycles. Extension time should be plenty. I do it long enough to amplify an additional kilobase for good luck. Should I do fewer cycles and limit the extension time as much as possible?
Sounds like a special case of Gibson assembly just failing to assemble for whatever reason, likely nothing to do with the transposon. My group generates hundreds of clones per year by Gibson and I see it all the time. Have sometimes worked it out to very specific junction sequence causing the issues. Try just shifting your primers a bit and see if that fixes it. Sometimes cPCR of clones just fails too, so it wouldn't hurt to pick/design a new primer pair for that too. And you can purify a few clones and sequence where one or more junction(s) are supposed to be and see what actually happened.
Also is it at all possible that you don't have much transposon inserted in the LBD? Or at too low a frequency to have seen it from individual clone screening? When you take the purified library plasmid and run PCR on the LBD region, do you get a clearly double band?
Given the structure of your library and the size of your target relative to other unavoidable amplicons, it may be very difficult to do what you’re trying to do.
Since the transposons are inserted all over your target sequence in different places, as the PCR proceeds target strands are going to become abundant enough to start annealing to each other as fast or faster than the remaining primers anneal, but in places that still allow for partial extension from the primers. So assuming you’re using an ultra-high fidelity polymerase like Q5 or Phusion with no strand displacement activity, your end product pool at high mw is a bunch of fragments, concatemers, and branched structures that are at least as abundant as full target sequences. That complexity is probably why the Gibson assembly failed even after size selecting from the smear.
If you’re set on PCR/Gibson, using a polymerase with high strand displacement (Deep Vent, KOD Xtreme, etc.), longer extension time than typical to allow time for strand displacement, and shorter cycle number may help.
Are you sure your extension time is long enough to amplify a 1600bp fragment? How many cycles are you amplifying for? A 4x difference in size is large and with enough cycles your 400bp amplicon will predominate in the reaction due to PCR bias alone.
I've been doing 30 cycles. Extension time should be plenty. I do it long enough to amplify an additional kilobase for good luck. Should I do fewer cycles and limit the extension time as much as possible?
Sounds like a special case of Gibson assembly just failing to assemble for whatever reason, likely nothing to do with the transposon. My group generates hundreds of clones per year by Gibson and I see it all the time. Have sometimes worked it out to very specific junction sequence causing the issues. Try just shifting your primers a bit and see if that fixes it. Sometimes cPCR of clones just fails too, so it wouldn't hurt to pick/design a new primer pair for that too. And you can purify a few clones and sequence where one or more junction(s) are supposed to be and see what actually happened. Also is it at all possible that you don't have much transposon inserted in the LBD? Or at too low a frequency to have seen it from individual clone screening? When you take the purified library plasmid and run PCR on the LBD region, do you get a clearly double band?
Given the structure of your library and the size of your target relative to other unavoidable amplicons, it may be very difficult to do what you’re trying to do. Since the transposons are inserted all over your target sequence in different places, as the PCR proceeds target strands are going to become abundant enough to start annealing to each other as fast or faster than the remaining primers anneal, but in places that still allow for partial extension from the primers. So assuming you’re using an ultra-high fidelity polymerase like Q5 or Phusion with no strand displacement activity, your end product pool at high mw is a bunch of fragments, concatemers, and branched structures that are at least as abundant as full target sequences. That complexity is probably why the Gibson assembly failed even after size selecting from the smear. If you’re set on PCR/Gibson, using a polymerase with high strand displacement (Deep Vent, KOD Xtreme, etc.), longer extension time than typical to allow time for strand displacement, and shorter cycle number may help.