pKK223-3 (1) contains the strong tac promoter upstream from the
multiple cloning site and the strong rrnB ribosomal terminator downstream
for control of protein expression (2-4).
Induction: tac promoter is inducible with 1-5 mM IPTG. Promoter is
very strong; even uninduced cells may show a low level of expression.
Expression: Genes containing a ribosome-binding site and ATG can be
inserted into any unique site in the MCS for expression. The ribosomebinding
site on the plasmid can be utilised to express inserts if the ATG
start codon of the insert is within 5-13 bp from the provided ribosomebindingsite (5, 6).
Transcription terminators: rrnB transcription terminators stabilize the
plasmid by inhibiting read-through transcription initiated from the tac
promoter in the parent plasmid.
Host(s): lac Iq strains; E. coli JM105 is recommended.
Selectable marker(s): Plasmid confers resistance to 100 μg/ml ampicillin.
Amplification: Recommended.
Control Regions: Expression control region: tac promoter: -10: 50-44; -
35: 72-67; Ribosome binding site: 11. MCS: 4552-4587. rrnB operon
region: 5S rRNA region: 4471-4352; rrnB T1 terminator: 4348-4305;
rrnB T2 terminator: 4173-4146. b-lactamase gene region: Promoter: -10:
4082-4077; -35: 4105-4100; Start codon (ATG): 4035; Stop codon
(TAA): 3178. Plasmid replication region: Site of replication initiation:
2417-2415; Region necessary for replication: 3111-2415.
