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A goal two minutes from time condemned Gillingham to defeat in their League 1 opener.
Birthday-boy Danny Kedwell hit the woodwork in an entertaining opening half that surprisingly failed to produce a goal.
The Gills bossed much of the second half but Colchester stepped it up in the latter stages and got their reward when Andrew Bond hit the winner.
Martin Allen threw in a surprise with his starting XI by naming transfer-listed Charlie Lee in midfield.
Lee’s only visible action of the pre-season was a 45-minute run-out against Faversham in June, while talks of a move to Southend dominated the rest of his summer.
Although he may not have had much involvement in pre-season, he was in the thick of it for Gills early on.
Gillingham set the tempo in the opening exchanges but just three minutes had gone when Matt Fish went down clutching his knee. He hobbled off, making way for teenager Stephen Butcher, who was making his first senior appearance.
It was to be a busy first half for him, as Colchester pumped balls into the right-back area, where big striker Jabo Ibehre and pacey winger Freddie Sears looked to make life difficult for the youngster.
Up front Allen chose to partner Kedwell - celebrating his 30 birthday - with summer signing Cody McDonald. It was Kedwell who was looking Gillingham’s biggest threat.
He smashed a ninth-minute effort narrowly wide, after Lee’s throw had caused panic in the box, and also crashed an effort off the woodwork, after spotting visiting goalkeeper Sam Walker off his line.
The Colchester stopper made a good save from Lee late in the half, while at the other end Stuart Nelson also had to make several timely interventions.
Despite plenty of action, the game was goalless at the break.
The Gills dictated the early stages of the second half but failed to make the most of a series of corners. With an hour gone Myles Weston was introduced to add extra pace to the attack, replacing McDonald, While Adebayo Akinfenwa came on moments later.
But it was a rare chance at the other end, for Colchester, that almost broke the deadlock as Nelson’s flying save denied Marcus Bean.
Chris Whelpdale’s low drive almost made the breakthrough, as Walker parried into the path of Amine Linganzi, but the linesman’s flag was raised for offside.
At the other end Nelson had to be alert to save from Ibehre but knew little of Alex Gilbey’s powerful header on 76 minutes, which he somehow saved. He held on to another Gilbey header with five minutes left.
But with two minutes remaining Colchester substitute Bond latched on to a low cross and drilled it home in front of the delirious 779 away fans.
Gillingham: Nelson, Fish (Butcher 4mins), Martin, Legge, Barrett, Gregory, Linganzi, Lee (Akinfenwa 62mins), Whelpdale, McDonald (Weston 56mins), Kedwell. Subs not used: Davies, Dack, German, Howard.
Colchester: Walker, David Wright, Dickson, Okuonghae, Eastman, Wilson, Massey, Eastmond (Bond 87mins), Sears (Drey Wright 78mins), Bean (Gilbey 74mins), Ibehre. Subs not used: Cousins, Thompson, Morrison, Olufemi.
Attendance: 6,792