Part:BBa_K5487114
MCS Box-lac operator-lac promoter-CAP binding site
M13 fwd-MCS-M13 rev Description and Function: M13 fwd and M13 rev: These are primer binding sites commonly used for sequencing or amplification of plasmid inserts. They flank the multiple cloning site (MCS), providing a convenient method for verifying the integrity of inserted DNA sequences. MCS (Multiple Cloning Site): This region contains a series of unique restriction sites that facilitate the easy insertion or removal of DNA fragments, allowing for versatile cloning strategies. Design Considerations: Sequence Integrity: Ensure that the MCS retains all its restriction sites without mutations, which can occur due to errors in DNA synthesis or during cloning. Spacer Regions: Include sufficient space between the M13 primer sites and the MCS to prevent steric hindrance during PCR or sequencing reactions. Orientation: Align the M13 primer sites correctly relative to the MCS to ensure straightforward and reliable sequencing of inserted fragments.
lac Operator and lac Promoter Description and Function: lac Promoter: A strong promoter that controls the expression of downstream genes in response to lactose or IPTG (Isopropyl β-D-1-thiogalactopyranoside), an analog of lactose that induces the lac operon. lac Operator: A specific DNA sequence within the lac promoter where the lac repressor binds, inhibiting transcription unless induced by IPTG. Design Considerations: Regulatory Dynamics: The placement of the lac operator should be close enough to the lac promoter to ensure effective repression in the absence of inducer and efficient activation upon induction. Promoter Strength: Modify the lac promoter, if necessary, to suit the expression levels required for the experiment. This might involve altering its sequence to increase or decrease its activity. Compatibility with Host Strain: Consider the host strain's background, as some strains may have mutations in the lac operon that affect the functionality of the lac promoter/operator system.
CAP Binding Site Description and Function: CAP Binding Site: A regulatory sequence located upstream of the lac promoter. The Catabolite Activator Protein (CAP) binds here when cAMP levels are high, enhancing the lac promoter's activity, hence facilitating more robust gene expression in the presence of IPTG. Design Considerations: Site Optimization: Ensure that the CAP binding site is correctly positioned relative to the lac promoter to maximize the synergistic effect of CAP binding on transcription enhancement. Environmental Conditions: Since CAP activity is influenced by glucose levels in the growth medium (affecting cAMP levels), consider the growth conditions under which the plasmid will be used to optimize expression levels.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 72
Illegal XbaI site found at 45
Illegal PstI site found at 33 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 72
Illegal PstI site found at 33 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 72
Illegal BamHI site found at 51 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 72
Illegal XbaI site found at 45
Illegal PstI site found at 33 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 72
Illegal XbaI site found at 45
Illegal PstI site found at 33 - 1000COMPATIBLE WITH RFC[1000]
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