Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Lorenzo Pasotti, Giacomo Zambianchi, Federica Sampietro, Paolo Magni
Date created: 2010-10-02 11:00:00
Date modified: 2015-05-08 01:11:52
[LoxP-KanMX-LoxP] excisable marker for yeast
| Types | DnaRegion |
| Roles | engineered_region Generator |
| Sequences | BBa_K300989_sequence (Version 1) |
Description
This cassette is to be used in yeast.It is composed by the kanMX gene from the E. coli transposon Tn903 that confers the resistance to the aminoglycoside antibiotic G418 to yeast (commonly used at 200 ug/ml working concentration). This gene is expressed by the TEF2 promoter/RBS and terminated by the TEF2 transcriptional terminator from Ashbya gossypii filamentous fungus.
The cassette is flanked by two loxP sites and for this reason it can be excised with Cre/loxP mediated marker removal recombination procedure.
ADVANTAGES IN GENOME ENGINEERING APPLICATIONS:
This part can be used to select positive integrants in the yeast genome and then it can be eliminated through Cre/loxP procedure. This enables the engineering of yeast genome in multiple steps, always using the same dominant selection marker.
Notes
The original sequence of loxP-KanMX-loxP from pUG6 vector has two NsiI restriction sites in kanR(Tn903) coding sequence and they have been removed in this part by changing codons.The TEF2 promoter has a XbaI and a PstI restriction sites, which have not been removed to avoid the change of the promoter strength. For this reason, this part is not fully compatible with RFC10 BioBrick Standard Assembly, but it can be assembled by using EcoRI-SpeI enzymes.
Source
pUG6 vector (GenBank: AF298793.1).| Sequence Annotation | Location | Component / Role(s) |
| BBa_J61046 (loxP) TEF2 (A. gossypii)+RBS kanR(Tn903) A->T T->C TEF2 terminator (A. gossypii) BBa_J61046 (loxP) kanMX cassette | 1,34 35,432 433,1242 708,708 975,975 1243,1507 1508,1541 35,1507 | feature/BioBrick engineered_region feature/promoter promoter CDS feature/cds point_mutation feature/s_mutation point_mutation feature/s_mutation feature/misc sequence_feature engineered_region feature/BioBrick sequence_feature feature/misc |