Notebook – Protocol – 1. Primer design

1.DNA sequence of E.coli adenylate cyclase (cyaA; accession code NC_000913 REGION: 3991153..3993699; 2547 bp) and E. coli IIA(Glc) (crr; accession code J02796; 510 bp) were obtained from Genebank

2.Both DNA sequences were checked for the “in availability” of restriction sites of EcoRI (G^AATT_C), XbaI (T^CTAG_A), SpeI (A^CTAG_T), and PstI (C_TGCA^G) by Bioedit program using menu: Sequence/Nucleic Acid/Restriction Map/Generate Map.

3. Forward primers were designed to contain (from 5’ direction) :

• additional sequence to accommodate restriction enzyme recognition; TTA
• sequence from backbone pSB1C3 which has both EcoRI and XbaI sites; GAATTCGCGGCCGCTTCTAGA
• seven codons of the initial part of the genes which include start codon

4. Reverse primers were designed to contain (from 5’ direction) :

• additional sequence to accommodate restriction enzyme recognition; TTA
• sequence from backbone pSB1C3 which has SpeI site; ACTAGTA
• double stop codon (in reverse complement sequence); TTATTA
• six codons of the last part of the genes (in reverse complement sequence)
• For cyaA, besides the last part of the genes which codes for full length sequence, other reverse primer was also designed to contain the last part of N-terminal catalytic domain (full length sequence, 2547 bp; N-terminal catalytic domain, 1605 bp)

Resulted primers sequences were as follow

• cyaA full length forward primer (primer name “ACEF”)
5’-TTA GAA TTC GCG GCC GCT TCT AGA TGT ACC TCT ATA TTG AGA CTC TG-3’
• cyaA catalytic domain reverse primer (primer name “ACER1”)
5’-TTA ACT AGT ATT ATT ATT CCG AGA GAT CGG GTG AAA T-3’
• cyaA full length reverse primer (primer name “ACER2”)
5’-TTA ACT AGT ATT ATT ACG AAA AAT ATT GCT GTA ATA G-3’
• crr forward primer (primer name “AEF”)
5’-TTA GAA TTC GCG GCC GCT TCT AGA TGG GTT TGT TCG ATA AAC TG-3’
• crr reverse primer (primer name “AER”)
5’-TTA ACT AGT ATT ATT ACT TCT TGA TGC GGA TAA C-3’

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