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From 2010.igem.org

Legend

• This color represents the portion to be added by Extension PCR / Inverse PCR / Assembly PCR
• This color represents sites that lie in the middle of the parts to be submitted. These are placed so that the promoters and the inserts can be switched out.
• This represents an apparent restriction site, one that cannot be used due to the sequences' presence in an ORF

The following were the primers we designed for conducting our experiments we obtained from other laboratories in our institute. For Monellin, we planned to use Single Chain Monellin obtained from primary literature as the gene-of-interest.
Gene of 295 bp to be synthesised by Assembly PCR / LCR.

Block 1

• Kpn1 | P170 | RBS | HindIII
• Kpn1 | NICE | RBS | HindIII
• HindIII| GFP | Terminator | BamH1
• Kpn1| Constitutive | RBS | GFP | Terminator | BamH1

Block 2

• Kpn1 | Constitutive | RBS | HindIII | SP310mut2 | Nhe1
• Nhe1 | GFP | Terminator | BamH1 (Extension PCR from 1.4

Block 3

• Kpn1 | Constitutive | RBS | HindIII
• HindIII | CRE-NLS | Terminator | Sac1
• Sac1 | Constitutive | RBS | loxP | Nhe1 | GFP | Terminator | loxP | Xho1 (by Inverse PCR + Extension PCR
• Xho1 | RFP | Terminator | BamH1

Final Design

• Kpn1 | NICE | RBS | HindIII (as made in 1.1)
• HindIII | CRE-NLS | Terminator | Sac1 (as made in 3.2)
• Sac1 | loxP | P170 | RBS | SP310mut2 | Xho1 (by Inverse PCR + Extension PCR)
• Xho1 | SCM | Nhe1 | loxP | Terminator | BamH1

Part Submission

EcoR1 | Not1 | Xba1 | NICE | RBS | Spe1 | Not1 | Pst1

EcoR1 | Not1 | Xba1 | P170 | RBS | Spe1 | Not1 | Pst1

EcoR1 | Not1 | Xba1 | Kpn1 | Constitutive | RBS | HindIII | SP310mut2 | Nhe1 | GFP | Terminator | BamH1 | Spe1 | Not1 | Pst1

EcoR1 | Not1 | Xba1 | Kpn1 | Constitutive | RBS | HindIII | CRE | Terminator | Sac1 | Constitutive | RBS | loxP | Sma1 | GFP | Terminator | loxP | Xho1 | RFP | Terminator | BamH1 | Spe1 | Not1 | Xba1

EcoR1 | Not1 | Xba1 | Kpn1 | NICE | RBS | HindIII | CRE-NLS | Terminator | Sac1 | loxP | P170 | RBS | SP310mut2 | Xho1 | SCM | BamH1 | loxP | Terminator | Spe1 | Not1 | Pst1

Proof of Concept

Block 1 - To characterize promoters against a standard

• P170 | GFP | Terminator
• NICE | GFP | Terminator
• Constitutive | GFP | Terminator

Block 2 - Test of export efficiency

• Constitutive | GFP | Terminator
• Constitutive | SP310mut2 | GFP | Terminator

Block 3 - To test CRE-loxP system

• Constitutive | RBS | CRE | Terminator | Constitutive | RBS | loxP | GFP | Terminator | loxP | RFP | Terminator :Acid Tolerance Promoter (P170) | CRE | Terminator | loxP | Consitutive | GFP | Terminator | loxP | RFP | Terminator

Final Proposed Design

NICE | CRE | Terminator | loxP | Acid Tolerance Inducer (P170) | Export tag | SCM | loxP | Terminator

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