pDsRed2-ER is a mammalian expression vector that encodes a fusion consisting of Discosoma sp. red fluorescent protein (DsRed2); the endoplasmic reticulum (ER) targeting sequence of calreticulin, fused to the 5 end of DsRed2; and the ER retention sequence, KDEL, fused to the 3 end of DsRed2a human codon-optimized DsRed variant engineered for faster maturation and lower non-specific aggregation.To drive expression of DsRed2, this vector contains the immediate early promoter of cytomegalovirus (PCMV IE). SV40 polyadenylation signals downstream of the DsRed2 gene direct proper processing of the 3-end of the DsRed2 mRNA transcript. The vector also contains an SV40 origin for replication in any mammalian cell line that expresses the SV40 T-antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin resistance cassetteconsisting of the SV40 early promoter (PSV40e), the neomycin/kanamycin resistance gene of Tn5 (Neor/Kanr), and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK poly A) geneallows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter (P) upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli.
| 载体基本信息 | |||
| 载体名称 | pDsRed2-ER | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 4757 bp | 载体类型 | Fluorescent Protein Reporter Vectors |
| 筛选标记 | Neomycin | 启动子 | CMV |
| 克隆方法 | Enzyme digestion and ligation | ||
pDsRed2-ER 载体载体图谱
pDsRed2-ER 载体载体序列
LOCUS Exported 4757 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 4757)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4757
/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"
CDS 663..1337
/codon_start=1
/product="improved tetrameric variant of DsRed fluorescent
protein"
/note="DsRed2"
/note="mammalian codon-optimized"
/translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
LFL"
misc_feature 1365..1421
/note="MCS"
/note="multiple cloning site"
polyA_signal 1545..1666
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(1673..2128)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2155..2259
/gene="bla"
/note="AmpR promoter"
promoter 2261..2618
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2469..2604
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2653..3447
/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 3679..3726
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4055..4643
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
