pDsRED-Monomer-N1

价格:1200元

联系方式:I47-825O-882O

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载体基本信息

出品公司: Clontech
载体名称: pDsRed-Monomer-N1
质粒类型: 哺乳动物细胞表达载体;荧光报告载体
高拷贝/低拷贝: 高拷贝
启动子: CMV
克隆方法: 多克隆位点,限制性内切酶
载体大小: 4691bp
5' 测序引物及序列: 5'd[GTACTGGAACTGGGGGGACAG]
3' 测序引物及序列: --
载体标签: 红色荧光蛋白DsRed-Monomer (C-term)
载体抗性: Kanamycin
筛选标记: Neomycin
克隆菌株: DH5α, HB101
宿主细胞(系): 常规细胞系,293、CV-1、CHO等
备注:
pDsRed-Monomer-N1载体是荧光报告载体,表达C端DsRed-Monomer融合蛋白;
DsRed-Monomer是DsRed的单体突变体,与DsRed相比,编码序列经过了人工优化,适用于在哺乳动物细胞中
的高水平表达。
产品目录号: 632465
稳定性: 稳表达 或 瞬表达
组成型: 组成型
病毒/非病毒: 非病毒

载体质粒图谱和多克隆位点信息

pDsRED-Monomer-N1载体图谱
pDsRED-Monomer-N1多克隆位点

pDsRED-Monomer-N1载体特征

载体简介

pDsRed-Express-N1载体描述

pDsRed-Express-N1 is a mammalian expression vector that encodes DsRed-Express, a variant of Discosoma sp. red fluorescent protein (DsRed; 1). DsRed-Express contains nine amino acid substitutions (listed on page 2), which improve the solubility of the protein and reduce the time from transfection to detection of red fluorescence (2). In addition, these substitutions reduce the level of residual green emission (2). When DsRed-Express is expressed in mammalian cell cultures, redemitting cells can be detected by either fluorescence microscopy or flow cytometry 8–12 hours after transfection (DsRed-Express excitation and emission maxima = 557 nm and 579 nm, respectively). Although DsRed-Express most likely forms the same tetrameric structure as wild-type DsRed, DsRed- Express displays a reduced tendency to aggregate (2). The DsRed-Express coding sequence is human codon-optimized for high expression in mammalian cells (3).
The multiple cloning site (MCS) in pDsRed-Express-N1 is positioned between the immediate early promoter of CMV (PCMV IE) and the DsRed-Express coding sequence. Genes cloned into the MCS are expressed as fusions to the N-terminus of DsRed-Express if they are in the same reading frame as DsRed-Express and there are no intervening stop codons. Sequences upstream of DsRed-Express have been converted to a Kozak consensus translation initiation site to increase translation efficiency in eukaryotic cells (4). SV40 polyadenylation signals downstream of the DsRed-Express gene direct proper processing of the 3' end of the DsRed-Express 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.


pDsRed-Express-N1 can be used to construct fusions to the N-terminus of DsRed-Express. If a fusion construct retains the fluorescent properties of the native DsRed-Express 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 pDsRed-Express-N1 so that it is in frame with the DsRed-Express coding sequence, with no intervening in-frame stop codons. The inserted gene should include an initiating ATG codon. Recombinant pDsRed-Express-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (5). pDsRed-Express-N1 can also be used as a cotransfection marker; the unmodified vector will express DsRed-Express.


pDsRed-Express-N1载体含有以下元件:

 Human cytomegalovirus (CMV) immediate early promoter: 1–589
Enhancer region: 59–465; TATA box: 554–560
Transcription start point: 583
C→G mutation to remove Sac I site: 569
 MCS: 591–671
 Discosoma sp. Red Fluorescent Protein (DsRed-Express) gene
Kozak consensus translation initiation site: 672–682
Start codon (ATG): 679–681; Stop codon: 1354–1356
CGC→GCC (Arg-2 to Ala) mutation: 682–684
AAG→GAG (Lys-5 to Glu) mutation: 691–693
AAC→GAC (Asn-6 to Asp) mutation: 694–696
ACC→TCC (Thr-21 to Ser) mutation: 739–741
CAC→ACC (His-41 to Thr) mutation: 799–801
AAC→CAG (Asn-42 to Gln) mutation: 802–804
GTG→GCC (Val-44 to Ala) mutation: 808–810
TGC→TCC (Cys-117 to Ser) mutation: 1027–1029
ACC→GCC (Thr-217 to Ala) mutation: 1327–1329
 SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1508–1513 & 1537–1542; mRNA 3' ends: 1546 & 1558
 f1 single-strand DNA origin: 1605–2060 (Packages the noncoding strand of DsRed-Express.)
 Bacterial promoter for expression of Kanr gene:
–35 region: 2122–2127; –10 region: 2145–2150
Transcription start point: 2157
 SV40 origin of replication: 2401–2536
 SV40 early promoter
Enhancer (72-bp tandem repeats): 2234–2305 & 2306–2377
21-bp repeats: 2381–2401, 2402–2422 & 2424–2444
Early promoter element: 2457–2463
Major transcription start points: 2453, 2491, 2497 & 2502
 Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences: Start codon (ATG): 2585–2587; Stop codon: 3377–3379
G→A mutation to remove Pst I site: 2767
C→A (Arg to Ser) mutation to remove BssH II site: 3113
 Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3615–3620 & 3628–3633
 pUC plasmid replication origin: 3964–4607
Sequencing primer locations
 DsRed1-N Sequencing Primer (Cat. No. 632387; 5'-GTACTGGAACTGGGGGGACAG-3'): 879–859

Propagation in E. coli
 Suitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requires
a host containing an F plasmid, such as the JM109 or XL1-Blue strains..
 Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts.
 E. coli replication origin: pUC
 Copy number: ≈500
 Plasmid incompatibility group: pMB1/ColE1

Excitation and emission maxima of DsRed-Express
 Excitation maximum = 557 nm
 Emission maximum = 579 nm

载体序列

LOCUS       pDsRed-Monomer-N1	4691 bp 	DNA	SYN
DEFINITION  pDsRed-Monomer-N1
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     source          1..4691
                     /organism="pDsRed-Monomer-N1"
                     /mol_type="other DNA"
     promoter        10..562
                     /label="CMV_immearly_promoter"
     misc_feature    65..352
                     /label="CAG_enhancer"
     misc_feature    519..539
                     /label="CMV_fwd_primer"
     promoter        520..589
                     /label="CMV_promoter"
     CDS             679..1356
                     /label="ORF frame 1"

     gene            682..1353
                     /label="DsRed_Monomer"
                     /gene="DsRed_Monomer"

     misc_feature    complement(859..879)
                     /label="dsRed1_N_primer"

     misc_feature    1570..1589
                     /label="EBV_rev_primer"

     rep_origin      complement(1739..2045)
                     /label="f1_origin"

     promoter        2124..2152
                     /label="AmpR_promoter"

     misc_feature    complement(2218..2238)
                     /label="pBABE_3_primer"

     misc_feature    complement(2224..2439)
                     /label="SV40_enhancer"

     promoter        2236..2504
                     /label="SV40_promoter"

     rep_origin      2403..2480
                     /label="SV40_origin"

     misc_feature    2465..2484
                     /label="SV40pro_F_primer"

     CDS             2587..3381
                     /label="ORF frame 1"

     gene            2590..3378
                     /label="NeoR/KanR"
                     /gene="NeoR/KanR"

     CDS             complement(2896..3432)
                     /label="ORF frame 3"

     terminator      3556..3825
                     /label="TK_PA_terminator"

     rep_origin      3973..4592
                     /label="pBR322_origin"

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 GGACAACACC GAGGACGTCA TCAAGGAGTT CATGCAGTTC
  721 AAGGTGCGCA TGGAGGGCTC CGTGAACGGC CACTACTTCG AGATCGAGGG CGAGGGCGAG
  781 GGCAAGCCCT ACGAGGGCAC CCAGACCGCC AAGCTGCAGG TGACCAAGGG CGGCCCCCTG
  841 CCCTTCGCCT GGGACATCCT GTCCCCCCAG TTCCAGTACG GCTCCAAGGC CTACGTGAAG
  901 CACCCCGCCG ACATCCCCGA CTACATGAAG CTGTCCTTCC CCGAGGGCTT CACCTGGGAG
  961 CGCTCCATGA ACTTCGAGGA CGGCGGCGTG GTGGAGGTGC AGCAGGACTC CTCCCTGCAG
 1021 GACGGCACCT TCATCTACAA GGTGAAGTTC AAGGGCGTGA ACTTCCCCGC CGACGGCCCC
 1081 GTAATGCAGA AGAAGACTGC CGGCTGGGAG CCCTCCACCG AGAAGCTGTA CCCCCAGGAC
 1141 GGCGTGCTGA AGGGCGAGAT CTCCCACGCC CTGAAGCTGA AGGACGGCGG CCACTACACC
 1201 TGCGACTTCA AGACCGTGTA CAAGGCCAAG AAGCCCGTGC AGCTGCCCGG CAACCACTAC
 1261 GTGGACTCCA AGCTGGACAT CACCAACCAC AACGAGGACT ACACCGTGGT GGAGCAGTAC
 1321 GAGCACGCCG AGGCCCGCCA CTCCGGCTCC CAGTAGAGCG GCCGCGACTC TAGATCATAA
 1381 TCAGCCATAC CACATTTGTA GAGGTTTTAC TTGCTTTAAA AAACCTCCCA CACCTCCCCC
 1441 TGAACCTGAA ACATAAAATG AATGCAATTG TTGTTGTTAA CTTGTTTATT GCAGCTTATA
 1501 ATGGTTACAA ATAAAGCAAT AGCATCACAA ATTTCACAAA TAAAGCATTT TTTTCACTGC
 1561 ATTCTAGTTG TGGTTTGTCC AAACTCATCA ATGTATCTTA AGGCGTAAAT TGTAAGCGTT
 1621 AATATTTTGT TAAAATTCGC GTTAAATTTT TGTTAAATCA GCTCATTTTT TAACCAATAG
 1681 GCCGAAATCG GCAAAATCCC TTATAAATCA AAAGAATAGA CCGAGATAGG GTTGAGTGTT
 1741 GTTCCAGTTT GGAACAAGAG TCCACTATTA AAGAACGTGG ACTCCAACGT CAAAGGGCGA
 1801 AAAACCGTCT ATCAGGGCGA TGGCCCACTA CGTGAACCAT CACCCTAATC AAGTTTTTTG
 1861 GGGTCGAGGT GCCGTAAAGC ACTAAATCGG AACCCTAAAG GGAGCCCCCG ATTTAGAGCT
 1921 TGACGGGGAA AGCCGGCGAA CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA AGGAGCGGGC
 1981 GCTAGGGCGC TGGCAAGTGT AGCGGTCACG CTGCGCGTAA CCACCACACC CGCCGCGCTT
 2041 AATGCGCCGC TACAGGGCGC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT
 2101 ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA
 2161 TAAATGCTTC AATAATATTG AAAAAGGAAG AGTCCTGAGG CGGAAAGAAC CAGCTGTGGA
 2221 ATGTGTGTCA GTTAGGGTGT GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA
 2281 GCATGCATCT CAATTAGTCA GCAACCAGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA
 2341 GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAT AGTCCCGCCC CTAACTCCGC
 2401 CCATCCCGCC CCTAACTCCG CCCAGTTCCG CCCATTCTCC GCCCCATGGC TGACTAATTT
 2461 TTTTTATTTA TGCAGAGGCC GAGGCCGCCT CGGCCTCTGA GCTATTCCAG AAGTAGTGAG
 2521 GAGGCTTTTT TGGAGGCCTA GGCTTTTGCA AAGATCGATC AAGAGACAGG ATGAGGATCG
 2581 TTTCGCATGA TTGAACAAGA TGGATTGCAC GCAGGTTCTC CGGCCGCTTG GGTGGAGAGG
 2641 CTATTCGGCT ATGACTGGGC ACAACAGACA ATCGGCTGCT CTGATGCCGC CGTGTTCCGG
 2701 CTGTCAGCGC AGGGGCGCCC GGTTCTTTTT GTCAAGACCG ACCTGTCCGG TGCCCTGAAT
 2761 GAACTGCAAG ACGAGGCAGC GCGGCTATCG TGGCTGGCCA CGACGGGCGT TCCTTGCGCA
 2821 GCTGTGCTCG ACGTTGTCAC TGAAGCGGGA AGGGACTGGC TGCTATTGGG CGAAGTGCCG
 2881 GGGCAGGATC TCCTGTCATC TCACCTTGCT CCTGCCGAGA AAGTATCCAT CATGGCTGAT
 2941 GCAATGCGGC GGCTGCATAC GCTTGATCCG GCTACCTGCC CATTCGACCA CCAAGCGAAA
 3001 CATCGCATCG AGCGAGCACG TACTCGGATG GAAGCCGGTC TTGTCGATCA GGATGATCTG
 3061 GACGAAGAGC ATCAGGGGCT CGCGCCAGCC GAACTGTTCG CCAGGCTCAA GGCGAGCATG
 3121 CCCGACGGCG AGGATCTCGT CGTGACCCAT GGCGATGCCT GCTTGCCGAA TATCATGGTG
 3181 GAAAATGGCC GCTTTTCTGG ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GGACCGCTAT
 3241 CAGGACATAG CGTTGGCTAC CCGTGATATT GCTGAAGAGC TTGGCGGCGA ATGGGCTGAC
 3301 CGCTTCCTCG TGCTTTACGG TATCGCCGCT CCCGATTCGC AGCGCATCGC CTTCTATCGC
 3361 CTTCTTGACG AGTTCTTCTG AGCGGGACTC TGGGGTTCGA AATGACCGAC CAAGCGACGC
 3421 CCAACCTGCC ATCACGAGAT TTCGATTCCA CCGCCGCCTT CTATGAAAGG TTGGGCTTCG
 3481 GAATCGTTTT CCGGGACGCC GGCTGGATGA TCCTCCAGCG CGGGGATCTC ATGCTGGAGT
 3541 TCTTCGCCCA CCCTAGGGGG AGGCTAACTG AAACACGGAA GGAGACAATA CCGGAAGGAA
 3601 CCCGCGCTAT GACGGCAATA AAAAGACAGA ATAAAACGCA CGGTGTTGGG TCGTTTGTTC
 3661 ATAAACGCGG GGTTCGGTCC CAGGGCTGGC ACTCTGTCGA TACCCCACCG AGACCCCATT
 3721 GGGGCCAATA CGCCCGCGTT TCTTCCTTTT CCCCACCCCA CCCCCCAAGT TCGGGTGAAG
 3781 GCCCAGGGCT CGCAGCCAAC GTCGGGGCGG CAGGCCCTGC CATAGCCTCA GGTTACTCAT
 3841 ATATACTTTA GATTGATTTA AAACTTCATT TTTAATTTAA AAGGATCTAG GTGAAGATCC
 3901 TTTTTGATAA TCTCATGACC AAAATCCCTT AACGTGAGTT TTCGTTCCAC TGAGCGTCAG
 3961 ACCCCGTAGA AAAGATCAAA GGATCTTCTT GAGATCCTTT TTTTCTGCGC GTAATCTGCT
 4021 GCTTGCAAAC AAAAAAACCA CCGCTACCAG CGGTGGTTTG TTTGCCGGAT CAAGAGCTAC
 4081 CAACTCTTTT TCCGAAGGTA ACTGGCTTCA GCAGAGCGCA GATACCAAAT ACTGTCCTTC
 4141 TAGTGTAGCC GTAGTTAGGC CACCACTTCA AGAACTCTGT AGCACCGCCT ACATACCTCG
 4201 CTCTGCTAAT CCTGTTACCA GTGGCTGCTG CCAGTGGCGA TAAGTCGTGT CTTACCGGGT
 4261 TGGACTCAAG ACGATAGTTA CCGGATAAGG CGCAGCGGTC GGGCTGAACG GGGGGTTCGT
 4321 GCACACAGCC CAGCTTGGAG CGAACGACCT ACACCGAACT GAGATACCTA CAGCGTGAGC
 4381 TATGAGAAAG CGCCACGCTT CCCGAAGGGA GAAAGGCGGA CAGGTATCCG GTAAGCGGCA
 4441 GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC TTCCAGGGGG AAACGCCTGG TATCTTTATA
 4501 GTCCTGTCGG GTTTCGCCAC CTCTGACTTG AGCGTCGATT TTTGTGATGC TCGTCAGGGG
 4561 GGCGGAGCCT ATGGAAAAAC GCCAGCAACG CGGCCTTTTT ACGGTTCCTG GCCTTTTGCT
 4621 GGCCTTTTGC TCACATGTTC TTTCCTGCGT TATCCCCTGA TTCTGTGGAT AACCGTATTA
 4681 CCGCCATGCA T
//

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