Primers for aiiA gene from Dr Fray.doc

aiiA gene from Dr Fray

ATGACAGTAAAGAAGCTTTATTTCGTACCAGCAGGTCGTTGTATGTTGGATCATTCGTCTGTTAACAGTA 70
CATTAACACGAGGGGAATTATTAGACTTACCGGTTTGGTGTTATCTTTTGGAGACTGAAGAAGGACCTAT 140
TTTAGTAGATACAGGTATGCCAGAAAGTGCGGTTAATAATGAAGGCCTTTTTAACGGTACATTTGTTGAA 210
GGACATATTTTACCAAAAATGACTGAAGAAGATAGAATCGTGAATATTTTAAAACGCGTTGGTTATGAGC 280
CGGAAGACCTTCTTTATATTATTAGTTCTCACTTACATTTTGATCATGCAGGAGGAAATGGTGCTTTTAC 350
AAATACACCGATTATTGTGCAGCGTGCTGAATACGAGGCGGCACAACATAGTGAAGAATATATGAAAGAA 420
TGTATATTGCCGAATTTGAAATACAAGATCATTGAAGGGGATTATGAAGTCGTACCAGGAGTTCAATTAT 490
TGTATACACCAGGACATACTCCGGGGCATCAGTCGCTATTAATTGAGACAGAAAAATCCGGTCCTGTGTT 560
ATTAACGATTGATGCATCTTATACGAAAGAAAATTTTGAAGATGAAGTGCCGTTCGCTGGATTTGATTCG 630
GAATTAGCTTTATCTTCAATTAAACGTTTAAAAGAAGTTGTTAGGAAAGAGAACCCAATTGTTTTCTTTG 700
GACATGATATAGAGCAGGAAAAGAGTTGTAAAGTGTTCCCTGAATATATATAG

Components we want to add

Degredation Tag + Stop codons

Gctgcaaacgacgaaaactacgctttagtagcttaataa

Epitope tag + start codon

ATGgattataaagatgatgatgataaaggt

Restriction sites

• Start

	- gaattcgcggccgcatctagag
		EcoRI		XbaI

• End

	- tactagtagcggccgctgcag
	    SpeI		   PstI

Desired Sequence

We are cutting with spe1 and ecoR1 so the 3’end can have the Pst1 restrection site removed to shorten the primers

Restriction Sites-Epitope tag-gene Degredation tag-Restriction Sites

GAATTCGCGGCCGCATCTAGAGATGGATTATAAAGATGATGATGATAAAGGTATGACAGTAAAGAAGCTT 70
TATTTCGTACCAGCAGGTCGTTGTATGTTGGATCATTCGTCTGTTAACAGTACATTAACACGAGGGGAAT 140
TATTAGACTTACCGGTTTGGTGTTATCTTTTGGAGACTGAAGAAGGACCTATTTTAGTAGATACAGGTAT 210
GCCAGAAAGTGCGGTTAATAATGAAGGCCTTTTTAACGGTACATTTGTTGAAGGACATATTTTACCAAAA 280
ATGACTGAAGAAGATAGAATCGTGAATATTTTAAAACGCGTTGGTTATGAGCCGGAAGACCTTCTTTATA 350
TTATTAGTTCTCACTTACATTTTGATCATGCAGGAGGAAATGGTGCTTTTACAAATACACCGATTATTGT 420
GCAGCGTGCTGAATACGAGGCGGCACAACATAGTGAAGAATATATGAAAGAATGTATATTGCCGAATTTG 490
AAATACAAGATCATTGAAGGGGATTATGAAGTCGTACCAGGAGTTCAATTATTGTATACACCAGGACATA 560
CTCCGGGGCATCAGTCGCTATTAATTGAGACAGAAAAATCCGGTCCTGTGTTATTAACGATTGATGCATC 630
TTATACGAAAGAAAATTTTGAAGATGAAGTGCCGTTCGCTGGATTTGATTCGGAATTAGCTTTATCTTCA 700
ATTAAACGTTTAAAAGAAGTTGTTAGGAAAGAGAACCCAATTGTTTTCTTTGGACATGATATAGAGCAGG 770
AAAAGAGTTGTAAAGTGTTCCCTGAATATATAGCTGCAAACGACGAAAACTACGCTTTAGTAGCTTAATA 840
ATACTAGTA

Primers

• 5’end = same as gene.

gaattcgcggccgcatctagagATGgattataaagatgatgatgataaaggtATGACAGTAAAGAAGCTTTATTTCG

• 3’end = reverse and complementary to gene

TACTAGTATTATTAAGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGCTATATATTCAGGGAACACTTTAC