BBa_K1833999 1 BBa_K1833999 T7 promoter as annealed oligos 2015-09-14T11:00:00Z 2015-09-16T07:58:33Z Synthesized oligonucleotides as described on main page. This is a part used as a building block for T7 driven expression. However, this part uses a special method with the following synthesized nucleotides: pT7sense: aattcgcggccgcttctagataatacgactcactatagggagaa pT7antisense: ctagttctccctatagtgagtcgtattatctagaagcggccgcg The following method can be used to insert a T7 promoter before a desired Biobrick part: 1. Cut the desired plasmid (which should contain an RBS and cds at minimum) 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. false false _2259_ 27388 27388 9 false When ligating with a cut plasmid, this leaves a Biobrick scar between the T7 promoter and original Biobrick part. false Konstantin Borisov annotation2460116 1 Oligo scar range2460116 1 24 29 annotation2460074 1 pT7 range2460074 1 1 23 BBa_K1833999_sequence 1 taatacgactcactatagggagaactaga igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 Chris J. Myers James Alastair McLaughlin 2017-03-06T15:00:00.000Z