I have transformed a drought-related gene of 1.7kb into pGEMt vector (E.coli DH5 alpha strain). After sequencing the plasmids (3 replicates) 4 changed amino acids are visible at different locations in all three replicates. How would I know that these different amino acids would affect the protein function or should I go ahead with them?
Read this and continue with your experiment
https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloning-tutorial/a/bacterial-transformation-selection
You don't use these plasmids. Sequence more and find one that does NOT have a missense mutation.
What is the method of sequence- Sanger sequencing. Sanger sequencing won't provide you correct sequence when reading more than 500bases. Try resequencing those plasmids again with new primers placed 500 bases apart from each other. As you said, your insert is 1.7kb, you may need at least 4 sequencing primers. And do verify for sequence by both forward and reverse reaction. Hope my suggestion will help you.
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