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in suitable host, produces protein 5-enolpyruvylshikimate-3-phosphate synthase

integration helper plasmid

shuttle vector

Construct size (kb): 180.0

DESCRIPTION OF VECTOR COMPONENT:
Name of vector: pMON530
Intact vector size:
Type of vector: plasmid
Vector end: BglII
Vector end: EcoRI
Cloning sites: BglII ClaI KpnI XhoI EcoRI
Polylinker sites: BglII ClaI KpnI XhoI EcoRI
Construction: pMON316 pMON526
Host range: Agrobacterium tumefaciens; Escherichia coli
Features (with orientation and position when available):
coding sequence: NOS, ->
marker(s): spcR
marker(s): strR
marker(s): kanR, <-
terminator: NOS polyadenylation signal, <-
MCS: EcoRI...BglII, ->
promoter: CaMV, <-
replicon: oriT
replicon: pMB1
Cross references:

Gene product: 5-enolpyruvylshikimate-3-phosphate synthase [EPSPS]

DESCRIPTION OF INSERT COMPONENT:
Genome: Petunia sp.
Gene symbol: EPSPS
Gene name: 5-enolpyruvylshikimate-3-phosphate synthase
Contains complete coding sequence?: Y
Tissue: MG4-G cell line
Type of DNA: cDNA
Insert end: EcoRI Modification: EcoRI -> BglII
Insert end: EcoRI
Insert size (kb): 1.95
Cross references:

Target Gene: , 5-enolpyruvylshikimate-3-phosphate synthase

A BglII+EcoRI digest produces two fragments from the insert. The approx. 330 bp BglII/EcoRI fragment contains 5' non-coding region and sequences encoding a chloroplast transit peptide and the 5' end of EPSPS.

The 1.62 kb EcoRI fragment encodes the 3' end of EPSPS and contains the poly(A) tail.

pMON546 contains a nopaline synthase/neomycin phosphotransferase II fusion gene that confers kanR only in transformed plant tissue.

Both pMON546 and pGV3111SE can be isolated from the A. tumefaciens culture by co-culturing with E. coli containing a helper mobilization plasmid like pRK2013. E. coli with pMON546 are spcR strR kanS, while E. coli with pGV311SE are spcS strS kanR.

pGV3111SE is a crippled Ti plasmid (the region containing tms tmr tml and OCS has been deleted) that contains vir genes which will integrate T-DNA sequences into a host chromosome.

When Petunia cells are transformed with A. tumifaciens containing pMON546 and pGV3111SE, the CaMV/EPSPS gene permits growth in the presence of 0.5mM glyphosate.

Phenotype verified: kanR, spcR, strR, cmlR.