Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Trinh Nguyen
Date created: 2016-10-18 11:00:00
Date modified: 2016-10-19 03:49:03
pENTR attB-flipped eYFP-attP (TP901)
| Types | DnaRegion |
| Roles | DNA sequence_feature |
| Sequences | BBa_K2100049_sequence (Version 1) |
Description
This basic part entry vector is flanked by L1 and L2 sites, which are used to denote a gene. This can be cascaded with a promoter (flanked by L4, R1 sites) using an LR reaction and cloned into a backbone that has a negative selection marker between R4 and R2 sites. This part adheres to RFC 65 for recombination based cloning of mammalian parts.The flipped eYFP gene is oriented upside down, preventing translation of the gene. The flipped fluorescent gene is flanked by recognition sites of TP901 recombinase, which is oriented in a way that only allows unidirectional flip. When recombinase TP901 is present, they will bind to the recognition sites and flip the upside-down eYFP to the correct orientation, allowing the fluorescent gene to be expressed.
Notes
We used Golden Gate reaction with BsaI restriction enzyme to assembly the parts into the backbone vector. We don't want to have BsaI sites within the eYFP sequence. Additionally, we had to consider how to oriented the Q sites in the extended regions of the PCR primers, so that the eYFP gene would be upside-down after annealing into the backbone.Source
Flipped eYFP was obtained from extension PCR from BBa_K2100005 (pENTR eYFP). The recognition sites of TP901 were ordered as single-single strands oligos from IDT and we got the sequences for the recognition sites from the BU Wetlab Team.| Sequence Annotation | Location | Component / Role(s) |
| flipped eYFP TP901 attB L1 TP901 attP L2 Kan(R) | 1362,2081 1308,1357 1176,1274 2099,2148 2176,2271 2442,3251 | feature/protein CDS feature/dna sequence_feature sequence_feature feature/dna feature/dna sequence_feature feature/dna sequence_feature feature/dna sequence_feature |