pmaxFP-Red-N vector

Vector description
pmaxFP-Red-N vector is an eukaryotic (mammalian) expression vector carrying maxFP-Red gene with humanized codon usage. maxFP-Red is a novel red fluorescent protein obtained by mutagenesis of Anthomedusae jellyfish chromoprotein. maxFP-Red is a non-aggregating tag which successful performance in fusions was demonstrated.
pmaxFP-Red-N is designed to generate fusion proteins for expression, in vivo localization, protein-protein interaction and co-localization studies or to express maxFP-Red protein in eukaryotic (mammalian) cells. pmaxFP-Red-N vector contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication and neomycin resistance (Neor) gene for selection in eukaryotic cells. It also contains pUC origin of replication for propagation in E. coli and f1 origin for single-stranded DNA production. Bacterial promoter (P) provides expression of kanamycin resistance gene in E. coli. To increase maxFP-Red translation, Kozak consensus translation initiation site is generated upstream of maxFP-Red sequence. Multiple cloning site (MCS) is between PCMVIE and maxFP-Red coding sequence.
Generation of maxFP-Red-fusion proteins A localization signal (or a gene of interest) should be cloned into MCS of the vector. It will be expressed as a fusion to maxFP-Red N-terminus when inserted in the same reading frame as maxFP-Red and no intervening stop codons are present. The inserted sequence should contain an initiating ATG codon.

Expression in mammalian cells
pmaxFP-Red-N can be transfected into mammalian cells by any known transfection method. If required, stable transformants can be selected. maxFP-Red-tagged fusions retain fluorescent properties of the native maxFP-Red protein allowing fusion protein localization in vivo.

Propagation in E. coli

• Suitable host strains: DH5alpha, HB101, and other general purpose strains. Single-stranded DNA production requires a host containing an F plasmid such as JM109 or XL1-Blue
• Selectable marker: plasmid confers resistance to kanamycin (30 µg/ml) to E. coli hosts
• E. coli replication origin: pUC
• Copy number: ~500
• Plasmid incompatibility group: pMB1/ColE1

Location of features:

PCMV IE: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
MCS: 591-671
maxFP-Red gene
Kozak consensus translation initiation site: 672-682
Start codon (ATG): 679-681; Stop codon: 1405-1407
Polyadenylation signals: 1561-1566 & 1590-1595
mRNA 3' ends: 1599 & 1611
f1 single-strand DNA origin: 1658-2113
(packages the noncoding strand of maxFP-Red)
Bacterial promoter expression of Kanr gene:
-35 region: 2175-2180; -10 region: 2198-2203
Transcription start point: 2210
SV40 origin of replication: 2454-2589
SV40 early promoter
Enhancer (72-bp tandem repeats): 2287-2358 & 2359-2430
21-bp repeats: 2434-2454, 2455-2475, & 2477-2497
Early promoter element: 2510-2516
Major transcription start points: 2506, 2544, 2550 & 2555
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2638-2640; stop codon: 3430-3432
G->A mutation to remove PstI site: 2820
C->A (Arg to Ser) mutation to remove BssHII site: 3166
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3668-3673 & 3681-3686
pUC plasmid replication origin: 4017-4660

*This product contains a propietary nucleic acid coding for a proprietary fluorescent protein(s) intended to be used for research purposes only. Any use of the proprietary nucleic acid or fluorescent proteins coding by proprietary nucleic acids other then for research use is strictly prohibited. USE IN ANY OTHER APPLICATION REQUIRES LICENSE FROM EVROGEN. To obtain such a license, please contact Evrogen at  license@evrogen.com.