pDsRED-Monomer-N1

pDsRED-Monomer-N1

pDsRED-Monomer-N1载体基本信息

pDsRED-Monomer-N1载体质粒图谱和多克隆位点信息

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

pDsRED-Monomer-N1载体序列

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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