Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena Buescher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Schaefer, Carolin Schmelas, Silvan Schmitz, Max Waldhauer
Date created: 2014-10-07 11:00:00
Date modified: 2015-05-08 01:10:04
RBS+NpuDnaE C-intein RFC[105] assembly construct (with His6)
| Types | DnaRegion |
| Roles | engineered_region Composite |
| Sequences | BBa_K1362101_sequence (Version 1) |
Description
This intein assembly construct is part of the iGEM team Heidelberg 2014's strategy for cloning with split inteins. Inteins are naturally occuring peptide sequences that splice out of a precursor protein and attach the remaining ends together to form a new protein. When splitting those intein sequence into an N-terminal and a C-terminal split intein one is left with a powerful tool to post-translationally modify whole proteins on the amino-acid sequence level. This construct was designed to express any protein of interest fused to the Nostoc punctiforme DnaE N-terminal split intein. The corresponding C-terminal construct isUpon coexpression or mixture of the N- and C-constructs protein splicing takes place and the N- and C-terminal proteins of interest are irreversibly assembled via a newly formed peptide bond.
This mechanism can be applied for a variety of different uses such as the activation of a protein through reconstitution of individually expressed split halves. See our split sfGFP experiment and the respective [[Part:BBa_K1362170|parts]] in the registry for more information. Protein splicing offers many new possibilities and we hope to have set a foundation that you guys can build on!
Nostoc punctiforme DnaE split intein (NpuDnaE)
Inteins can be found in all kingdoms of life, however their use for the organism remains yet unknown. In the past years many intein sequences were found in the genomes of cyanobacteria.
NpuDnaE is a naturally split intein from the alpha subunit of Dna Polymerase III in Nostoc punctiforme bacteria which shows [[#References|[2]]] extraordinarly high splicing activity and therefore is on of the gold-standards for inteins since many years.
Amino Acid Sequence:
N-Intein: AEY | CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN
C-Intein: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN | CFN
Notes
???Source
The part is assembled from different sources. The intein sequences come addgene or were kindly provided by different working groups. BBa_J04450 was used as mRFP selection marker. The scars and overhangs are part of the RFC[???] intein cloning strategy and are further explained on our wiki and in RFC[???] [[#Reference|[1]]]. Please see the corresponding basic parts for the individual detailed information.| Access | Instance | Definition |
| public public public public public public public public public public public | BBa_K1362090 BBa_G0000 BBa_K1362414 BBa_K1362431 BBa_K1362418 BBa_K1362464 BBa_K1362401 BBa_K1362419 BBa_K1362422 BBa_K1362421 BBa_K1362465 | T7 RBS scar RFC105 A His6 RFC105 CN 2/3Gly NpuDnaE(C) RFC105 CC mRFP sel. RFC105 Z AA |