Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Tom Geddes
Date created: 2014-10-06 11:00:00
Date modified: 2015-05-08 01:10:13
aeBlue-aacC1 gene cassette for integron testing
| Types | DnaRegion |
| Roles | engineered_region Reporter |
| Sequences | BBa_K1388001_sequence (Version 1) |
Description
This part is used to produce circular gene cassettes for integron testing. It contains (in order):- A spacer region designed to allow circularisation primers to anneal (this avoids secondary structure problems caused by attC, see below)
- An NheI restriction site
- An attC integron recombination site
- An aeBlue gene (see BBa_K864401) with RBS
- BamHI restriction site
- An aacC1 gene with RBS (confers resistance to gentamicin).
Cassettes can be produced by using Xba/SpeI or NheI/SpeI ELAN (cutting at the prefix/suffix or and ligating each end of the part together at these points) or using Gibson assembly; Gibson circularisation can be carried out by running PCR using primers with reverse-complementary 5 prime ends and 3' ends which anneal respectively to the spacer region and the end of aacC1. Examples of circularisation primers given below:
F: 5'-GAGGTCGGACAGTATATTGAAACGTCAGGAGTCTAGATACCGAGGGACAGACTACCAACTCACA-3'
R: 5'-GTATCTAGACTCCTGACGTTTCAATATACTGTCCGACCTCTTATTAGGTGGCGGTACTTGGGTCG-3'
Notes
- attC placed at beginning of part to allow for the option of cointegration of plasmids using integron recombination (retaining aeBlue/aacC1 near attI site and any nearby promoters).- 5' spacer region included to allow for primer annealing; attC has significant secondary structure which can make primer design difficult.
- NheI was placed to allow ELAN circularisation without SpeI without including unnecessary spacer region.
- BamHI was placed to allow excision of aacC1 gene with BamHI/Spe digest.
Source
Synthesised as a complete gBlock gene fragment from Integrated DNA Technologies: attC sequence was sourced from pUS23 and aacC1 was sourced from pUCP24. aeBlue sequence was taken from part BBa_K864401.| Sequence Annotation | Location | Component / Role(s) |
| Circularisation spacer region attC recombination site rbs (aeBlue) aeBlue rbs (aacC1) aacC1 (GmR) | 1,27 34,93 95,102 110,808 817,823 831,1298 | primer_binding_site feature/primer_binding feature/misc sequence_feature ribosome_entry_site feature/rbs CDS feature/cds feature/rbs ribosome_entry_site feature/cds CDS |