Given the gene sequence ATGATCGTTTTAGTAACTGGAGCAACGGCAGGTTTTGGTGAATGCATTACTCGTCG

Question

Given the gene sequence

ATGATCGTTTTAGTAACTGGAGCAACGGCAGGTTTTGGTGAATGCATTACTCGTCGTTTTATTCAACAAG
GGCATAAAGTTATCGCCACTGGCCGTCGCCAGGAACGGTTGCAGGAGTTAAAAGACGAACTGGGAGATAA
TCTGTATATCGCCCAACTGGACGTTCGCAACCGCGCCGCTATTGAAGAGATGCTGGCATCGCTTCCTGCC
GAGTGGTGCAATATTGATATCCTGGTAAATAATGCCGGCCTGGCGTTGGGCATGGAGCCTGCGCATAAAG
CCAGCGTTGAAGACTGGGAAACGATGATTGATACCAACAACAAAGGCCTGGTATATATGACGCGCGCCGT
CTTACCGGGTATGGTTGAACGTAATCATGGTCATATTATTAACATTGGCTCAACGGCAGGTAGCTGGCCG
TATGCCGGTGGTAACGTTTACGGTGCGACGAAAGCGTTTGTTCGTCAGTTTAGCCTGAATCTGCGTACGG
ATCTGCATGGTACGGCGGTGCGCGTCACCGACATCGAACCGGGTCTGGTGGGTGGTACCGAGTTTTCCAA
TGTCCGCTTTAAAGGCGATGACGGTAAAGCAGAAAAAACCTATCAAAATACCGTTGCATTGACGCCAGAA
GATGTCAGCGAAGCCGTCTGGTGGGTGTCAACGCTGCCTGCTCACGTCAATATCAATACCCTGGAAATGA
TGCCGGTTACCCAAAGCTATGCCGGACTGAATGTCCACCGTCAGTAA

Design forward and reverse primers for cloning the above gene in pET28a cloning vector (a) with a N-terminal 6xHis tag and (b) without any tag (just the protein).

pET28 cloning vector is given as (His tag highlighted in blue)

TITAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGT GCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGAAT TCGAGCTCCGTCGACAAGCITTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCC GGCTGCTAA

Explanation / Answer

In both, the case reverse primer will be same as we don't need His-tag as C-terminal.

With His-Tag-

Fw primer - 5' CATATGATGATCGTTTTAGTAACTG 3'

Reverse primer - 5' CTCGAGTTACTGACGGTGGACATTC 3'

Without His-Tag-

Forward primer - 5 ' CCATGGATGATCGTTTTAGTAACTG 3'

Reverse primer - 5' CTCGAGTTACTGACGGTGGACATTC 3'

the restriction site is bold and underlined

Related Questions

Navigate

• Browse (All)
• Browse G
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.