pDsRed2- N1 Plasmid

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pDsRed2-N1

 

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pDsRed2-N1,Plasmid pDsRed2-N1,pDsRed2-N1 vector

 

 

pDsRed2-N1 Information

Promoter: CMV promoter

Replicator: pUC ori, F1 ori

Terminator: SV40 poly (A) signal

Plasmid classification: lactation serial plasmids; lactating fluorescent plasmids; lactating red plasmids

Plasmid size: 4689bp

Plasmid label: C-DsRed2

Prokaryotic resistance: kanamycin Kan (50 mu g/ml)

Screening markers: neomycin Neo/G418

Cloned strains of Escherichia coli, DH5 A and other Escherichia coli

Culture conditions: 37 centigrade, aerobic LB

Expression host: mammalian cells such as 293T

Culture conditions: 37 C, 5%CO2

Induction mode: no induction, instantaneous expression

5'sequencing primers: CMV-F (CGCAAATGGGCGGTAGGCGTG)

3'sequencing primers: Sv40-polyA-R (GAAATTTGTGATGCTATTGC)

Remark: the red fluorescent expression vector of mammalian cell

 

pDsRed2-N1 Description

PDsRed2-N1 encoding DsRed2 is a DsRed variant designed for faster maturation and lower nonspecific aggregation. Derived from Discosoma sp. Red fluorescent protein DsRed2, such as its progenitor cell DsRed1, contains a series of silent base pairs, which correspond to the high expression of human codons in mammalian cells. In addition to these changes, DsRed2 contains six amino acid substitutions: V105A, I161T and S197A, which lead to the rapid development of red fluorescence in transfected cell lines; and R2A, K5E and K9T, which prevent protein aggregation. (DsRed2 may form the same four dimer structure with DsRed1). In group DsRed2 mammalian cell cultures, red blood cells could be detected within 24 hours after transfection. The insoluble aggregates of proteins frequently observed in DsRed1 and mammalian cell systems were significantly reduced in cells expressing DsRed2. More mature, more soluble red fluorescent protein was well tolerated by host cells; the mammalian cell cultures transfected with DsRed2 did not show significant signs of reduced viability. In those cells tested, the cells expressing DsRed2 showed the same form as non transfected controls (such as adhesion, refraction) and birth. Long features.

pDsRed2-N1 encodes DsRed2, a DsRed variant that has been engineered for faster maturation and lower non-specific aggregation. Derived from the Discosoma sp. red fluorescent protein (drFP583; 1), DsRed2, like its progenitor DsRed1, contains a series of silent base-pair changes that correspond to human codon-usage preferences for high expression in mammalian cells (2). In addition to these changes, DsRed2 contains six amino acid substitutions: V105A, I161T, and S197A, which result in the more rapid appearance of red fluorescence in transfected cell lines; and R2A, K5E, and K9T, which prevent the protein from aggregating. (DsRed2 may, however, form the same tetrameric structure as DsRed1 [3].) In mammalian cell cultures when DsRed2 is expressed constitutively, red-emitting cells can be detected by fluorescence microscopy within 24 hours of transfection. Large insoluble aggregates of protein, often observed in bacterial and mammalian cell systems expressing DsRed1, are dramatically reduced in cells expressing DsRed2. The faster-maturing, more soluble red fluorescent protein is also well tolerated by host cells; mammalian cell cultures transfected with DsRed2 show no obvious signs of reduced viability—in those cell lines tested, cells expressing DsRed2 display the same morphology (e.g., adherence, light-refraction) and growth characteristics as non-transfected controls. The multiple cloning site (MCS) in pDsRed2-N1 is positioned between the immediate early promoter of CMV (PCMV IE) and the DsRed2 coding sequence. Genes cloned into the MCS are expressed as fusions to the N-terminus of DsRed2. Sequences upstream of DsRed2 have been converted to a Kozak consensus translation initiation site to increase translation efficiency in eukaryotic cells (4). SV40 polyadenylation signals ownstream of the DsRed2 gene direct proper processing of the 3' end of the DsRed2 mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing 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 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. pDsRed2-N1 can be used to construct fusions to the N-terminus of DsRed2. If a fusion construct retains the fluorescent properties of the native DsRed2 protein, its expression can be monitored by flow cytometry and its localization in vivo can be determined by fluorescence microscopy. The target gene should be cloned into pDsRed2-N1 so that it is in frame with the DsRed2 coding sequence, with no intervening in-frame stop codons. The inserted gene should include an initiating ATG codon. Recombinant pDsRed2-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (5). Unmodified pDsRed2-N1 can also be used to express DsRed2 in a cell line of interest (e.g., for use as a transfection marker).

pDsRed2-N1-vector

 

pDsRed2-N1 Sequence

LOCUS       Exported                4689 bp ds-DNA    circular SYN 18-10-2015
KEYWORDS    Untitled 4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4689)
  AUTHORS   admin
  TITLE     Direct Submission
  JOURNAL   Exported 2015-10-18    
FEATURES             Location/Qualifiers
     source          1..4689
                     /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..1356
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /note="DsRed2"
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     polyA_signal    1477..1598
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1605..2060)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2087..2191
                     /gene="bla"
                     /note="AmpR promoter"
     promoter        2193..2550
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      2401..2536
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             2585..3379
                     /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    3611..3658
                     /note="HSV TK poly(A) signal"
                     /note="herpesvirus thymidine kinase polyadenylation signal"
     rep_origin      3987..4575
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccaccgg tcgccaccat ggcctcctcc gagaacgtca tcaccgagtt catgcgcttc
      721 aaggtgcgca tggagggcac cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
      781 ggccgcccct acgagggcca caacaccgtg aagctgaagg tgaccaaggg cggccccctg
      841 cccttcgcct gggacatcct gtccccccag ttccagtacg gctccaaggt gtacgtgaag
      901 caccccgccg acatccccga ctacaagaag ctgtccttcc ccgagggctt caagtgggag
      961 cgcgtgatga acttcgagga cggcggcgtg gcgaccgtga cccaggactc ctccctgcag
     1021 gacggctgct tcatctacaa ggtgaagttc atcggcgtga acttcccctc cgacggcccc
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