pIRES2-DsRed-Express contains the internal ribosome entry site (IRES; 1, 2) of the encephalomyocarditis virus (ECMV) between the MCS and a mutant of the Discosoma sp. red fluorescent protein DsRed-Express coding region (3–5). This permits both the gene of interest (cloned into the MCS) and the DsRed-Express gene to be translated from a single bicistronic mRNA. pIRES2-DsRed-Express is designed for the efficient selection by flow cytometry of transiently transfected mammalian cells expressing DsRed-Express and the protein of interest. This vector can also be used to express DsRed-Express alone or to obtain stably transfected cell lines without timeconsuming drug and clonal selection.DsRed-Express is a human codon-optimized (4) DsRed variant of Discosoma sp. red fluorescent protein (6). Its coding sequence contains nine amino acid substitutions which improve solubility of the protein and reduce the time from transfection to detection of red fluorescence (exitation and emission maxima = 557 nm and 579 nm, respectively). In addition, these substitutions reduce the level of residual green emission (5). Although DsRed-Express most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express displays a reduced tendency to aggregate (3, 5).The MCS in pIRES2-DsRed-Express is between the immediate early promoter of cytomegalovirus (P CMV IE ) and the IRES sequence. SV40 polyadenylation signals downstream of the DsRed-Express gene direct proper processing of the 3 end of the bicistronic mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neo r ), consisting of the SV40 early promoter, the neomycin/ kanamycin resistance gene of Tn5, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pIRES2-DsRed-Express backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
| 载体基本信息 | |||
| 载体名称 | pIRES2-DsRed-Express | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 5264 bp | 载体类型 | Mammalian Expression Vectors |
| 筛选标记 | Neomycin | 拷贝数 | High copy number |
| 启动子 | CMV | 克隆方法 | Enzyme digestion and ligation |
| 5引物 | CMV-F: CGCAAATGGGCGGTAGGCGTG | 3引物 | IRES-R: CCTCACATTGCCAAAAGACG |
pIRES2-DsRed-Express 载体载体图谱
pIRES2-DsRed-Express 载体载体序列
LOCUS Exported 5264 bp ds-DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5264)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5264
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 61..364
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 365..568
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
misc_feature 609..665
/note="MCS"
/note="multiple cloning site"
misc_feature 667..1253
/note="IRES2"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 1254..1931
/codon_start=1
/product="rapidly maturing tetrameric variant of DsRed
fluorescent protein (Bevis and Glick, 2002)"
/note="DsRed-Express"
/note="mammalian codon-optimized"
/translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
LFL"
polyA_signal 2052..2173
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(2180..2635)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2662..2766
/gene="bla"
/note="AmpR promoter"
promoter 2768..3125
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2976..3111
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 3160..3954
/codon_start=1
/gene="aph(3)-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 4186..4233
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4562..5150
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
