BBa_K1833997

BBa_K1833997 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K1833997
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Konstantin Borisov
Date created: 2015-09-15 11:00:00
Date modified: 2015-09-16 07:59:14

T3 promoter as annealed oligonucleotides



Types
DnaRegion

Roles
promoter

Regulatory

Sequences BBa_K1833997_sequence (Version 1)

Description

This is a part used as a building block for T3 driven expression. However, this part uses a special cloning method with the following synthesized nucleotides:


pT3sense: aattcgcggccgcttctagaaattaaccctcactaaagggagaa

pT3antisense: ctagttctccctttagtgagggttaatttctagaagcggccgcg


The following method can be used to insert pProt before a desired Biobrick part (which should contain an RBS at minimum and preferably a cds and terminator):

1. Cut the desired plasmid with EcoRI and XbaI. Purify the plasmid with a method of your choice (both gel purification and PCR purification kits from Qiagen serve to remove the short undesired oligonucleotide from the digestion).

2. Be sure that your oligos have 5' phosphorylation (treat unphosphorylated oligos with T4 Polynucleotide Kinase). Then, anneal the two oligos by slowly cooling from 98C to room temperature.

3. Finally, ligate the cut plasmid with the annealed oligos by using a 1:10 molar ratio of plasmid to insert.

4. Transform the ligation reaction into E. coli, and plate.

5. Pick colonies, and verify the success of the cloning with sequencing or PCR. (Doing a diagnostic digest is not recommended as the size of the original plasmid and the desired plasmid are very similar.)

6. Add a composite part to the iGEM registry by using this part with the original protein coding part, checking the box to create without a scar, as this part contains a modified scar.

Notes

When ligating with a cut plasmid, this leaves a modified Biobrick scar between the T7 promoter and original Biobrick part, which is included in the sequence of this part.

Source

Synthesized oligonucleotides as described on main page.

Sequence Annotation Location Component / Role(s)
pT3
Oligo scar
1,23
24,29
feature/promoter promoter
feature/scar
igem#status
Planning
 
synbiohub#ownedBy
user/james
 
synbiohub#ownedBy
user/myers
 
synbiohub#topLevel
BBa_K1833997/1