| 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 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
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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