Strong promoter + Strong RBS + CRYAB + 10x Histidine tag + Double terminator
the cDNA of cryab was ordered from OriGene. The promoter and rbs was from iGEM distribution kit (BBa_K880005) and His-Term was from an intermediate part we constructed (BBa_K1898500).
BBa_K880005 is used to provide a strong promoter and strong rbs to maximize protein output. The ORF includes cryab, a crystallin protein exists in the lens and other tissues, and 10x Histidine tag for protein purification. BBa_B0015 consists of a double terminator to end transcription.
We made sure that there was no Ecor1, Xbal, Spel1, and Pst1 cutting sites in the DNA. Primers were designed to remove the stop codon from cryab and were synthesized by Tri-I. Our assembly method BioBrick RFC[10] shifted the open reading frame and created a stop codon in between cryab and 10x histidine tag. The sequence was sent to Missiontech to add two base pairs in between the two genes.
Strong promoter, strong RBS combination for high expression levels of proteins
It is a combination of strong promoter (J23100) and RBS (B0034).
-J23100+B0034 -Strong promoter, strong RBS combination for high expression levels of proteins Consensus constitutive promoter and RBS sequence-produces strongest possible expression.
Josh Atkinson, Mike Ferguson, and Ben Parker
the cDNA or cryab is ordered from OriGene.
This gene codes for cryab, or crystallin alpha B, a crystallin protein exists in the lens and also other tissues and organs.
We made sure that there was no Ecor1, Xbal, Spel1, and Pst1 cutting sites in the DNA. Primers were designed to remove the stop codon and were synthesized by Tri-I biotech.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Double terminator consisting of BBa_B0010 and BBa_B0012
10x-Histidine (10x-His) Tag with double stop codon (TAATAA)
Constructed through oligonucleotide annealing
10x-Histidine tag with double stop codon TAATAA to allow for better extraction of tagged products and protein termination in a single part.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1