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Some history on the Gadsden flag, all of which can be found at http://www.gadsden.info/history.html
It’s not known exactly when the snake image and the phrase “Don’t Tread On Me” were joined together, but in 1775, Marines (joining the Navy on their first mission to capture British cargo ships loaded with arms) were seen carrying drums painted bright yellow with the snake logo and phrase printed on them.
Then, in December 1775, “An American Guesser” wrote to the Pennsylvania Journal
“I observed on one of the drums belonging to the marines now raising, there was painted a Rattle-Snake, with this modest motto under it, ‘Don’t tread on me.’ As I know it is the custom to have some device on the arms of every country, I supposed this may have been intended for the arms of America.”
This anonymous writer, having “nothing to do with public affairs” and “in order to divert an idle hour,” speculated on why a snake might be chosen as a symbol for America.
He suggested that “the Rattle-Snake is found in no other quarter of the world besides America.”
The rattlesnake also has sharp eyes, and “may therefore be esteemed an emblem of vigilance.”
“She never begins an attack, nor, when once engaged, ever surrenders: She is therefore an emblem of magnanimity and true courage. … she never wounds ’till she has generously given notice, even to her enemy, and cautioned him against the danger of treading on her.”
“I confess I was wholly at a loss what to make of the rattles, ’till I went back and counted them and found them just thirteen, exactly the number of the Colonies united in America; and I recollected too that this was the only part of the Snake which increased in numbers…
“‘Tis curious and amazing to observe how distinct and independent of each other the rattles of this animal are, and yet how firmly they are united together, so as never to be separated but by breaking them to pieces. One of those rattles singly, is incapable of producing sound, but the ringing of thirteen together, is sufficient to alarm the boldest man living.”
Many scholars now agree that this “American Guesser” was Benjamin Franklin.
Franklin is also known for opposing the use of an eagle — “a bird of bad moral character” — as a national symbol.
So, Franklin helped create the rattlesnake symbol, but the flag is usually referenced to Christopher Gadsden. Gadsden led the Sons of Liberty in South Carolina starting in 1765. He was a colonel in the Continental Army, was in Philadelphia in 1775 representing his home state of SC, and was on the Marine Committee who decided to outfit and man the Alfred ship (one of the ships acquired from the British as mentioned earlier) and its sister ships.
Esek Hopkins was chosen as commander-in-chief of the Navy, Gadsden presented him with the flag, and John Paul Jones put it up the flag pole.
Gadsden later presented the flag to the South Carolina legislature in Charleston, as recorded here:
“Col. Gadsden presented to the Congress an elegant standard, such as is to be used by the commander in chief of the American navy; being a yellow field, with a lively representation of a rattle-snake in the middle, in the attitude of going to strike, and these words underneath, “Don’t Tread on Me!”