pDsRed-Express-C1

pDsRed-Express-C1

pDsRed-Express-C1载体基本信息

pDsRed-Express-C1载体质粒图谱和多克隆位点信息

pDsRed-Express-C1载体描述

pDsRed-Express-C1 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, 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-C1 is positioned between the DsRed-Express coding sequence and the SV40 polyadenylation signal (SV40 poly A). Genes cloned into the MCS will be expressed as fusions to the C-terminus of DsRed-Express if they are in the same reading frame as DsRed-Express and there are no intervening stop codons. A Kozak consensus translation initiation site upstream of DsRed-Express increases the translation efficiency in eukaryotic cells (4). SV40 poly A signals downstream of the MCS direct proper processing of the 3' end of mRNA transcripts. The vector backbone also 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)—consisting 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—allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli.

pDsRed-Express-C1 can be used to construct fusions to the C-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-C1 so that it is in frame with the DsRed-Express coding sequences, with no intervening in-frame stop codons. The recombinant DsRed-Express vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (5). pDsRed-Express-C1 can also be used as a cotransfection marker; the unmodified vector will express DsRed-Express.

pDsRed-Express-C1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

 Discosoma sp. human codon-optimized Red Fluorescent Protein (DsRed-Express) gene

Kozak consensus translation initiation site: 606–616

Start codon (ATG): 613–615; Stop codon: 1357–1359

CGC→GCC (Arg-2 to Ala) mutation: 616–618

AAG→GAG (Lys-5 to Glu) mutation: 625–627

AAC→GAC (Asn-6 to Asp) mutation: 628–630

ACC→TCC (Thr-21 to Ser) mutation: 673–675

CAC→ACC (His-41 to Thr) mutation: 733–735

AAC→CAG (Asn-42 to Gln) mutation: 736–738

GTG→GCC (Val-44 to Ala) mutation: 742–744

TGC→TCC (Cys-117 to Ser) mutation: 961–963

ACC→GCC (Thr-217 to Ala) mutation: 1261–1263

Last amino acid in DsRed-Express: 1285–1287

 MCS: 1288–1344

 SV40 early mRNA polyadenylation signal

Polyadenylation signals: 1498–1503 & 1527–1532; mRNA

3' ends: 1536 & 1648

 f1 single-strand DNA origin: 1595–2050 (Packages the noncoding strand of DsRed-Express.)

 Bacterial promoter for expression of Kanr gene

–35 region: 21

12–2117; –10 region: 2135–2140

Transcription start point: 2147

 SV40 origin of replication: 2391–2526

 SV40 early promoter

Enhancer (72-bp tandem repeats): 2224–2295 & 2296–2367

21-bp repeats: 2371–2991, 2392–2412 & 2414–2431

Early promoter element: 2447–2453

Major transcription start points: 2443, 2481, 2487 & 2492

 Kanamycin/neomycin resistance gene

Neomycin phosphotransferase coding sequences:

Start codon (ATG): 2576–2578; stop codon: 3368–3370

G→A mutation to remove Pst I site: 2758

C→A (Arg to Ser) mutation to remove BssH II site: 3104

 Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal

Polyadenylation signals: 3606–3611 & 3619–3624

 pUC plasmid replication origin: 3955–4598

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 JM109 or XL1-Blue.

 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/Col E1

Excitation and emission maxima of DsRed-Express

 Excitation maximum = 557 nm

 Emission maximum = 579 nm

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 CCGGTCGCCA CCATGGCCTC CTCCGAGGAC GTCATCAAGG AGTTCATGCG CTTCAAGGTG

  661 CGCATGGAGG GCTCCGTGAA CGGCCACGAG TTCGAGATCG AGGGCGAGGG CGAGGGCCGC

  721 CCCTACGAGG GCACCCAGAC CGCCAAGCTG AAGGTGACCA AGGGCGGCCC CCTGCCCTTC

  781 GCCTGGGACA TCCTGTCCCC CCAGTTCCAG TACGGCTCCA AGGTGTACGT GAAGCACCCC

  841 GCCGACATCC CCGACTACAA GAAGCTGTCC TTCCCCGAGG GCTTCAAGTG GGAGCGCGTG

  901 ATGAACTTCG AGGACGGCGG CGTGGTGACC GTGACCCAGG ACTCCTCCCT GCAGGACGGC

  961 TCCTTCATCT ACAAGGTGAA GTTCATCGGC GTGAACTTCC CCTCCGACGG CCCCGTAATG

 1021 CAGAAGAAGA CTATGGGCTG GGAGGCCTCC ACCGAGCGCC TGTACCCCCG CGACGGCGTG

 1081 CTGAAGGGCG AGATCCACAA GGCCCTGAAG CTGAAGGACG GCGGCCACTA CCTGGTGGAG

 1141 TTCAAGTCCA TCTACATGGC CAAGAAGCCC GTGCAGCTGC CCGGCTACTA CTACGTGGAC

 1201 TCCAAGCTGG ACATCACCTC CCACAACGAG GACTACACCA TCGTGGAGCA GTACGAGCGC

 1261 GCCGAGGGCC GCCACCACCT GTTCCTGAGA TCTCGAGCTC AAGCTTCGAA TTCTGCAGTC

 1321 GACGGTACCG CGGGCCCGGG ATCCACCGGA TCTAGATAAC TGATCATAAT CAGCCATACC

 1381 ACATTTGTAG AGGTTTTACT TGCTTTAAAA AACCTCCCAC ACCTCCCCCT GAACCTGAAA

 1441 CATAAAATGA ATGCAATTGT TGTTGTTAAC TTGTTTATTG CAGCTTATAA TGGTTACAAA

 1501 TAAAGCAATA GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCA TTCTAGTTGT

 1561 GGTTTGTCCA AACTCATCAA TGTATCTTAA CGCGTAAATT GTAAGCGTTA ATATTTTGTT

 1621 AAAATTCGCG TTAAATTTTT GTTAAATCAG CTCATTTTTT AACCAATAGG CCGAAATCGG

 1681 CAAAATCCCT TATAAATCAA AAGAATAGAC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG

 1741 GAACAAGAGT CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA

 1801 TCAGGGCGAT GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG

 1861 CCGTAAAGCA CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA

 1921 GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT

 1981 GGCAAGTGTA GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT

 2041 ACAGGGCGCG TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT

 2101 TTTCTAAATA CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA

 2161 ATAATATTGA AAAAGGAAGA GTCCTGAGGC GGAAAGAACC AGCTGTGGAA TGTGTGTCAG

 2221 TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA GTATGCAAAG CATGCATCTC

 2281 AATTAGTCAG CAACCAGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA

 2341 AGCATGCATC TCAATTAGTC AGCAACCATA GTCCCGCCCC TAACTCCGCC CATCCCGCCC

 2401 CTAACTCCGC CCAGTTCCGC CCATTCTCCG CCCCATGGCT GACTAATTTT TTTTATTTAT

 2461 GCAGAGGCCG AGGCCGCCTC GGCCTCTGAG CTATTCCAGA AGTAGTGAGG AGGCTTTTTT

 2521 GGAGGCCTAG GCTTTTGCAA AGATCGATCA AGAGACAGGA TGAGGATCGT TTCGCATGAT

 2581 TGAACAAGAT GGATTGCACG CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC TATTCGGCTA

 2641 TGACTGGGCA CAACAGACAA TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA

 2701 GGGGCGCCCG GTTCTTTTTG TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAAGA

 2761 CGAGGCAGCG CGGCTATCGT GGCTGGCCAC GACGGGCGTT CCTTGCGCAG CTGTGCTCGA

 2821 CGTTGTCACT GAAGCGGGAA GGGACTGGCT GCTATTGGGC GAAGTGCCGG GGCAGGATCT

 2881 CCTGTCATCT CACCTTGCTC CTGCCGAGAA AGTATCCATC ATGGCTGATG CAATGCGGCG

 2941 GCTGCATACG CTTGATCCGG CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA

 3001 GCGAGCACGT ACTCGGATGG AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA

 3061 TCAGGGGCTC GCGCCAGCCG AACTGTTCGC CAGGCTCAAG GCGAGCATGC CCGACGGCGA

 3121 GGATCTCGTC GTGACCCATG GCGATGCCTG CTTGCCGAAT ATCATGGTGG AAAATGGCCG

 3181 CTTTTCTGGA TTCATCGACT GTGGCCGGCT GGGTGTGGCG GACCGCTATC AGGACATAGC

 3241 GTTGGCTACC CGTGATATTG CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT

 3301 GCTTTACGGT ATCGCCGCTC CCGATTCGCA GCGCATCGCC TTCTATCGCC TTCTTGACGA

 3361 GTTCTTCTGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC CAACCTGCCA

 3421 TCACGAGATT TCGATTCCAC CGCCGCCTTC TATGAAAGGT TGGGCTTCGG AATCGTTTTC

 3481 CGGGACGCCG GCTGGATGAT CCTCCAGCGC GGGGATCTCA TGCTGGAGTT CTTCGCCCAC

 3541 CCTAGGGGGA GGCTAACTGA AACACGGAAG GAGACAATAC CGGAAGGAAC CCGCGCTATG

 3601 ACGGCAATAA AAAGACAGAA TAAAACGCAC GGTGTTGGGT CGTTTGTTCA TAAACGCGGG

 3661 GTTCGGTCCC AGGGCTGGCA CTCTGTCGAT ACCCCACCGA GACCCCATTG GGGCCAATAC

 3721 GCCCGCGTTT CTTCCTTTTC CCCACCCCAC CCCCCAAGTT CGGGTGAAGG CCCAGGGCTC

 3781 GCAGCCAACG TCGGGGCGGC AGGCCCTGCC ATAGCCTCAG GTTACTCATA TATACTTTAG

 3841 ATTGATTTAA AACTTCATTT TTAATTTAAA AGGATCTAGG TGAAGATCCT TTTTGATAAT

 3901 CTCATGACCA AAATCCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAA

 3961 AAGATCAAAG GATCTTCTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTGCAAACA

 4021 AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC AAGAGCTACC AACTCTTTTT

 4081 CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG ATACCAAATA CTGTCCTTCT AGTGTAGCCG

 4141 TAGTTAGGCC ACCACTTCAA GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGCTAATC

 4201 CTGTTACCAG TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGA

 4261 CGATAGTTAC CGGATAAGGC GCAGCGGTCG GGCTGAACGG GGGGTTCGTG CACACAGCCC

 4321 AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC AGCGTGAGCT ATGAGAAAGC

 4381 GCCACGCTTC CCGAAGGGAG AAAGGCGGAC AGGTATCCGG TAAGCGGCAG GGTCGGAACA

 4441 GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGTCGGG

 4501 TTTCGCCACC TCTGACTTGA GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA

 4561 TGGAAAAACG CCAGCAACGC GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT

 4621 CACATGTTCT TTCCTGCGTT ATCCCCTGAT TCTGTGGATA ACCGTATTAC CGCCATGCAT

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