pDsRed1-N1 encodes a novel red fluorescent protein (RFP; 1) that has been optimized for high expression in mammalian cells (excitation maximum = 558 nm; emission maximum = 583 nm). RFP was isolated from an IndoPacific sea anemone-relative, Discosoma sp; DsRed1’s coding sequence contains 144 silent base pair changes, which correspond to human codon-usage preferences for high expression in mammalian cells (2). Sequences upstream of DsRed1 have been converted to a Kozak consensus translation initiation site (3) to increase translation efficiency in eukaryotic cells. The MCS is between the immediate early promoter of CMV (PCMV IE) and the DsRed1 coding sequence. Genes cloned into the MCS as described below are expressed as fusions to the N-terminus of DsRed1. SV40 polyadenylation signals downstream of the DsRed1 gene direct proper processing of the 3 end of the DsRed1 mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neor ) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists 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. A bacterial promoter upstream of the cassette confers kanamycin resistance to E. coli. The pDsRed1-N1 backbone also has a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.Fusions to the N terminus of DsRed1 typically do not alter the fluorescence properties of native DsRed1, allowing in vivo localization of the fusion protein.The target gene should be cloned into pDsRed1-N1 in frame with the DsRed1 coding sequence, with no intervening in-frame stop codons. The inserted gene should include an initiating ATG codon. Recombinant pDsRed1-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (4). Unmodified pDsRed1-N1 can also be used to express DsRed1 in a cell line of interest (e.g., for use as a transfection marker).
| 载体基本信息 | |||
| 载体名称 | pDsRed1-N1 | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 4692 bp | 载体类型 | Fluorescent Protein Reporter Vectors |
| 筛选标记 | Neomycin | 启动子 | CMV |
pDsRed1-N1 载体载体图谱
pDsRed1-N1 载体载体序列
LOCUS Exported 4692 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 4692)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4692
/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 591..671
/note="MCS"
/note="multiple cloning site"
CDS 679..1359
/codon_start=1
/product="wild-type DsRed"
/note="DsRed1"
/note="mammalian codon-optimized"
/translation="MVRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTV
KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
VVTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
KALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRH
HLFL"
polyA_signal 1480..1601
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(1608..2063)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2090..2194
/gene="bla"
/note="AmpR promoter"
promoter 2196..2553
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2404..2539
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2588..3382
/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 3614..3661
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 3990..4578
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
