I would like to use PCR to amplify just the coding sequence of a gene. I would also like to add an EcoR1 restriction site upstream of the start codon of the ORF. How can I find the upstream primer? Where is the start codon? And where would the EcoR1 restriction site be located?
The primers I plan to use are:
T T A A A G G C A A C A G C G C G A A C
T A G C G A A G G C G T A T T T T G C G
Below is the nucleic acid sequence for the gene of interest.
GAATATAGCTTTCCGACCCTGAAAGAAATTACCGGCAGCCTGGTGATTTATTATGTGAAA GGCACCAAAACCCTGGGCCTGTATTTTCCGAACCTGACCATTATTCGCGGCCAGACCCTG GCGTATAACTATGCGGTGGTGATTAAAGAAACCAACCTGCTGGTGATTAACCTGTTTCAG TATATTGGCCTGGTGAACCTGCGCCTGGTGATGTTTGGCGGCGTGCGCATTGATAACAAC CCGAGCCTGTGCTATGTGAACACCATTAACTGGACCAGCATTGTGCGCCAGCATGGCGCG CCGATTAAACTGATTAACATGCGCAACTTTTGCCTGGAATTTTGCCCGAACAACTGCCCG CGCGGCGAAAACCTGCAGAAATATTGCTGGCATCATAACACCTGCCAGAACGTGTGCAGC GAAAGCTGCGCGAAAAACAACCAGTATTGCTTTATGAACGATCCGAACCGCTGCTGCCAT CCGGAATGCCTGGGCGGCTGCTATGGCGAAGGCGATGAAATGTGCGTGAGCTGCAAAAAC GTGCTGAGCAACGGCCGCTGCGTGAAAAAATGCGATAAATTTTTTTATAAATATAAAAAC CGCCGCTGCATTAGCGAAAGCGAATGCATGAACCTGAGCAGCCAGAGCAGCACCGATTAT CGCCGCGAAGATCATTATGCGATTGAAGATGATGCGTGCGTGAAACAGTGCAGCCCGGGC AAACAGCAGAACAAAATTACCGGCCGCTGCGAAATTTGCGGCAGCGAATGCAGCAAAAAA TATTGCGGCCGCGTGCTGATTCATAGCATTAACGATTTTAGCGAAGCGGAAGGCTGCTAT AGCATTGAAGATATTTATATTAGCATTAAAGAAGGCGGCAGCAGCCTGAGCGATATTCTG ATGAAAGCGTTTAGCAAACTGACCATTATTGAAAACAGCCTGCGCATTGTGAAAAGCAGC AGCATTACCAGCCTGGATTTTCTGCGCAGCCTGCGCATTATTAAAGGCAACAGCGCGAAC AAAAACAACAACGAAACCACCAACGGCAGCAACAAAAGCATTGTGTTTGAAGTGTATGAT AACGAAAACCTGGTGGAAATTTTTGATGAAAAAGTGGCGCTGAACCAGCTGAACATTACC ATTCTGGAAGGCAGCGTGAGCTTTAACCTGAACCGCAAACTGTGCCCGGAAAAAATTCTG AGCTTTCTGAACAGCAACGTGCAGACCAAAACCAAACTGAGCAAACTGGATGAACGCATT ATTAACGTGAGCAACGGCGATATGGCGTTTTGCAAACATGAAAAACTGATTGTGCAGGTG CTGAACATTAGCGATAGCAGCTTTACCGTGAAATGCCATAGCAAAGTGCCGGATATTAGC CTGCAGAACAGCGCGCTGACCGCGCTGTTTATGCTGAAAGAAACCACCAAAAACGTGAGC CAGTATGAACTGTTTAACAGCTGCAGCGAACAGAACTGGCGCACCGTGGAAGGCTATATT GGCAAACCGGTGGTGATTGATAACCTGCTGCCGGCGAAACGCTATGCGTTTTATGCGGAA GCGATTCGCAGCCGCGTGGAAAACTGGCTGGAAATTAGCGATGTGATTTATCTGACCACC GAACGCAAAAACCCGAGCCAGCCGATTAACCTGAAAACCGAAACCCTGAGCAGCAGCGAA ATTCGCCTGACCTGGAACCCGCCGCTGTTTCCGAACGGCGAAGTGACCCGCTATATTATT TGGTATCGCAGCCTGGAAATTTTTGGCGATGATTTTTTTATGGAAGATGTGTGCTTTACC AAAAGCAAAAGCCAGCGCCGCATTCAGAGCCAGAGCAACAACCGCATTAGCAAAACCAAC AACGGCAACCAGAACCAGGGCGAATGCGATTGCGATAAATGCATGGAAAGCTGCAACAAA AACCAGATTCTGATTAACATTGATAAAGATGATACCATTATGCAGTTTCTGGATGAATAT ATTACCAAAAGCTTTATTAAACAGAACGAAATTCCGAAAAGCAACTGGAAACCGGTGGAT AGCGGCCATTTTGAAATTAAAAAAGTGGAATATAACCAGACCGTGCGCGTGGATGAAGTG GTGACCAACCTGCCGGCGACCGATATTAGCACCATTAGCAAACCGAGCATTGAAAACCTG ACCAACCATTTTAAAAAAATGGTGCAGGTGCCGGCGAACGAAACCTATTTTAAATTTACC AACCTGCATCATTATACCGAATATTTTTTTGAAATTCAGGCGTGCCAGGATCTGCAGAAC GTGCAGAGCAGCGAAAGCAAAGCGCTGAGCAAAAACGGCACCAGCAAAAACGTGCTGAAC CTGCCGTGCAGCAGCAAAAGCCTGATTAGCGCGCATACCAGCCCGAAAGAAAACATGGAT AACATTAACGAAAGCACCATTAACACCCGCGTGAACGGCAGCAGCGTGAAAATTTTTTGG AGCGAACCGAAAAGCCCGAACGGCTTTATTCTGAACTATTTTATTCGCTATCGCAAATAT CCGTATGAAGCGGAACTGGATTGGCTGAGCACCCCGTGCGTGACCCGCCCGGAATGGCTG AACGGCACCAACATTATGGCGAAAAACAGCAGCAACAGCACCAACATTATTCAGCAGAAA AGCGGCTTTTATACCCTGCGCGATCTGAAACCGGGCAAATATGAATTTCAGATTAAACCG TTTGCGATTGCGACCGAAGGCGCGTGGACCAAAAGCTATCATTTTAAAATTAGCAGCGAT AGCATTTTTACCGAATATCGCAACTATTTTATTATTGGCGCGGTGGTGATTATTATTATT CTGTGCGCGCTGTTTGTGCTGGTGTGCTATTATTGCAAAAAAAAATATCTGGAACGCACC CGCTGGGTGAGCAGCAACCCGGATTATTGGATTATTTATGAAATTGATGTGTGGGAAATT GATCGCAAAGATGTGGATATGCTGGAATGGAACTATCCGCTGGGCCGCGGCAGCTTTGGC ATGGTGTATAAAGGCGTGCTGAAAAACCTGACCACCCCGGCGCGCGATAAATATCTGCGC ACCCTGGATGAAAAACAGATGTTTGTGGCGGTGAAAACCCTGAACACCAACAGCACCATT CTGGAACGCCGCGAATTTATTAACGAAGCGTGCTATATGAAACAGTTTCAGAGCTATCAT CTGGTGAAACTGTATGGCATTGTGAGCAAA
