pDsRed-Monomer-N In-Fusion Ready

pDsRed-Monomer-N In-Fusion Ready载体基本信息

pDsRed-Monomer-N In-Fusion Ready载体质粒图谱和多克隆位点信息

pDsRed-Monomer-N In-Fusion Ready载体描述

pDsRed-Monomer-N In-Fusion Ready vector is a linearized mammalian expression vector that encodes DsRed-Monomer (DsRed. M1), a monomeric mutant derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed (1). DsRed-Monomer contains a total of forty-five amino acid substitutions. When DsRed-Monomer is expressed in mammalian cell cultures, red fluorescent cells can be detected by either fluorescence microscopy or flow cytometry 12–16 hr after transfection. DsRed-Monomer has an excitation maximum at 557 nm and an emission maximum at 592 nm. The DsRed-Monomer coding sequence is human codon-optimized for high expression levels in mammalian cells (2).The linearized vector allows direct cloning of PCR products without any need for restriction digestion when using the In-Fusion HD Cloning Plus (638909). This is accomplished by the use of a specific 15 nucleotide long sequence within the 5’ ends of the sense and antisense primers that overlap with the cut ends of the linearized vector.

The primers that will be used to amplify In-Fusion Ready PCR products require the following 15 nucleotides on their 5’ends:

Sense primer: 5’ AAGGCCTCTGTCGAC followed by sequence of amplification target 3’

Antisense primer: 5’ AGAATTCGCAAGCTT followed by sequence of amplification target 3’

If the sequence of the gene of interest is added in-frame immediately after the 15 nucleotides mentioned above, this sequence will automatically be in-frame with the DsRed-Monomer sequence downstream and therefore be expressed as a fusion protein to the N-terminus of DsRed-Monomer.

A Kozak consensus translation initiation site upstream of DsRed-Monomer increases the translation efficiency in eukaryotic cells (3). SV40 polyadenylation signals downstream of the DsRed-Monomer gene direct proper processing of the 3' end of the mRNA. transcript. The vector backbone contains an SV40 origin of 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 selection of stably transfected eukaryotic cells using G418. This cassette contains 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 allows the plasmid to confer kanamycin resistance in E. coli.

Fusions to the N-terminus of DsRed-Monomer retain the fluorescent properties of the native protein and allow monitoring of the fusion protein localization in vivo. The PCR-amplified gene of interest is directly cloned into the pDsRed-Monomer-N In-Fusion Ready Vector so that it is in-frame with the DsRed-Monomer coding sequences with no intervening in-frame stop codons. This can be accomplished by using the suggested primers. The inserted gene must include the initiating ATG codon. This recombinant vector can be transfected into mammalian cells using any standard transfection method. Stable transformants can be selected using G418 (2).

The DsRed1-N Sequencing Primer (Cat. No. 632387) can be used to sequence genes cloned adjacent to the 5’ end of the DsRed-Monomer coding region.

For Western blotting, the Living Colors DsRed Polyclonal Antibody (Cat. No. 632496) can be used to recognize the DsRed-Monomer protein. For optimal results for immunoprecipitation, we suggest you titrate the amount of antibody needed for quantitative recovery of the antigen. As a starting point, however, we recommend using the antibody at a 1:1,000 dilution.

Propagation in E. coli

 Suitable host strains: Stellar Competent Cells.

 Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.

 E. coli replication origin: pUC

 Copy number: ~ 500

 Plasmid incompatibility group: pMB1/ColE1

Excitation and emission maxima of DsRed-Monomer

 Excitation maximum = 557 nm

 Emission maximum = 592 nm

pDsRed-Monomer-N In-Fusion Ready载体序列

ORIGIN

    1 AGCTTGCGAA TTCTGGCCTA CCGGTCGCGG ACAACACCGA GGACGTCATC AAGGAGTTCA

   61 TGCAGTTCAA GGTGCGCATG GAGGGCTCCG TGAACGGCCA CTACTTCGAG ATCGAGGGCG

  121 AGGGCGAGGG CAAGCCCTAC GAGGGCACCC AGACCGCCAA GCTGCAGGTG ACCAAGGGCG

  181 GCCCCCTGCC CTTCGCCTGG GACATCCTGT CCCCCCAGTT CCAGTACGGC TCCAAGGCCT

  241 ACGTGAAGCA CCCCGCCGAC ATCCCCGACT ACATGAAGCT GTCCTTCCCC GAGGGCTTCA

  301 CCTGGGAGCG CTCCATGAAC TTCGAGGACG GCGGCGTGGT GGAGGTGCAG CAGGACTCCT

  361 CCCTGCAGGA CGGCACCTTC ATCTACAAGG TGAAGTTCAA GGGCGTGAAC TTCCCCGCCG

  421 ACGGCCCCGT AATGCAGAAG AAGACTGCCG GCTGGGAGCC CTCCACCGAG AAGCTGTACC

  481 CCCAGGACGG CGTGCTGAAG GGCGAGATCT CCCACGCCCT GAAGCTGAAG GACGGCGGCC

  541 ACTACACCTG CGACTTCAAG ACCGTGTACA AGGCCAAGAA GCCCGTGCAG CTGCCCGGCA

  601 ACCACTACGT GGACTCCAAG CTGGACATCA CCAACCACAA CGAGGACTAC ACCGTGGTGG

  661 AGCAGTACGA GCACGCCGAG GCCCGCCACT CCGGCTCCCA GTAGGCGGCC GCGACTGCGA

  721 CTCTAGATCA TAATCAGCCA TACCACATTT GTAGAGGTTT TACTTGCTTT AAAAAACCTC

  781 CCACACCTCC CCCTGAACCT GAAACATAAA ATGAATGCAA TTGTTGTTGT TAACTTGTTT

  841 ATTGCAGCTT ATAATGGTTA CAAATAAAGC AATAGCATCA CAAATTTCAC AAATAAAGCA

  901 TTTTTTTCAC TGCATTCTAG TTGTGGTTTG TCCAAACTCA TCAATGTATC TTAAGGCGTA

  961 AATTGTAAGC GTTAATATTT TGTTAAAATT CGCGTTAAAT TTTTGTTAAA TCAGCTCATT

 1021 TTTTAACCAA TAGGCCGAAA TCGGCAAAAT CCCTTATAAA TCAAAAGAAT AGACCGAGAT

 1081 AGGGTTGAGT GTTGTTCCAG TTTGGAACAA GAGTCCACTA TTAAAGAACG TGGACTCCAA

 1141 CGTCAAAGGG CGAAAAACCG TCTATCAGGG CGATGGCCCA CTACGTGAAC CATCACCCTA

 1201 ATCAAGTTTT TTGGGGTCGA GGTGCCGTAA AGCACTAAAT CGGAACCCTA AAGGGAGCCC

 1261 CCGATTTAGA GCTTGACGGG GAAAGCCGGC GAACGTGGCG AGAAAGGAAG GGAAGAAAGC

 1321 GAAAGGAGCG GGCGCTAGGG CGCTGGCAAG TGTAGCGGTC ACGCTGCGCG TAACCACCAC

 1381 ACCCGCCGCG CTTAATGCGC CGCTACAGGG CGCGTCAGGT GGCACTTTTC GGGGAAATGT

 1441 GCGCGGAACC CCTATTTGTT TATTTTTCTA AATACATTCA AATATGTATC CGCTCATGAG

 1501 ACAATAACCC TGATAAATGC TTCAATAATA TTGAAAAAGG AAGAGTCCTG AGGCGGAAAG

 1561 AACCAGCTGT GGAATGTGTG TCAGTTAGGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC

 1621 AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA GGTGTGGAAA GTCCCCAGGC

 1681 TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CATAGTCCCG

 1741 CCCCTAACTC CGCCCATCCC GCCCCTAACT CCGCCCAGTT CCGCCCATTC TCCGCCCCAT

 1801 GGCTGACTAA TTTTTTTTAT TTATGCAGAG GCCGAGGCCG CCTCGGCCTC TGAGCTATTC

 1861 CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC CTAGGCTTTT GCAAAGATCG ATCAAGAGAC

 1921 AGGATGAGGA TCGTTTCGCA TGATTGAACA AGATGGATTG CACGCAGGTT CTCCGGCCGC

 1981 TTGGGTGGAG AGGCTATTCG GCTATGACTG GGCACAACAG ACAATCGGCT GCTCTGATGC

 2041 CGCCGTGTTC CGGCTGTCAG CGCAGGGGCG CCCGGTTCTT TTTGTCAAGA CCGACCTGTC

 2101 CGGTGCCCTG AATGAACTGC AAGACGAGGC AGCGCGGCTA TCGTGGCTGG CCACGACGGG

 2161 CGTTCCTTGC GCAGCTGTGC TCGACGTTGT CACTGAAGCG GGAAGGGACT GGCTGCTATT

 2221 GGGCGAAGTG CCGGGGCAGG ATCTCCTGTC ATCTCACCTT GCTCCTGCCG AGAAAGTATC

 2281 CATCATGGCT GATGCAATGC GGCGGCTGCA TACGCTTGAT CCGGCTACCT GCCCATTCGA

 2341 CCACCAAGCG AAACATCGCA TCGAGCGAGC ACGTACTCGG ATGGAAGCCG GTCTTGTCGA

 2401 TCAGGATGAT CTGGACGAAG AGCATCAGGG GCTCGCGCCA GCCGAACTGT TCGCCAGGCT

 2461 CAAGGCGAGC ATGCCCGACG GCGAGGATCT CGTCGTGACC CATGGCGATG CCTGCTTGCC

 2521 GAATATCATG GTGGAAAATG GCCGCTTTTC TGGATTCATC GACTGTGGCC GGCTGGGTGT

 2581 GGCGGACCGC TATCAGGACA TAGCGTTGGC TACCCGTGAT ATTGCTGAAG AGCTTGGCGG

 2641 CGAATGGGCT GACCGCTTCC TCGTGCTTTA CGGTATCGCC GCTCCCGATT CGCAGCGCAT

 2701 CGCCTTCTAT CGCCTTCTTG ACGAGTTCTT CTGAGCGGGA CTCTGGGGTT CGAAATGACC

 2761 GACCAAGCGA CGCCCAACCT GCCATCACGA GATTTCGATT CCACCGCCGC CTTCTATGAA

 2821 AGGTTGGGCT TCGGAATCGT TTTCCGGGAC GCCGGCTGGA TGATCCTCCA GCGCGGGGAT

 2881 CTCATGCTGG AGTTCTTCGC CCACCCTAGG GGGAGGCTAA CTGAAACACG GAAGGAGACA

 2941 ATACCGGAAG GAACCCGCGC TATGACGGCA ATAAAAAGAC AGAATAAAAC GCACGGTGTT

 3001 GGGTCGTTTG TTCATAAACG CGGGGTTCGG TCCCAGGGCT GGCACTCTGT CGATACCCCA

 3061 CCGAGACCCC ATTGGGGCCA ATACGCCCGC GTTTCTTCCT TTTCCCCACC CCACCCCCCA

 3121 AGTTCGGGTG AAGGCCCAGG GCTCGCAGCC AACGTCGGGG CGGCAGGCCC TGCCATAGCC

 3181 TCAGGTTACT CATATATACT TTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC

 3241 TAGGTGAAGA TCCTTTTTGA TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC

 3301 CACTGAGCGT CAGACCCCGT AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG

 3361 CGCGTAATCT GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG

 3421 GATCAAGAGC TACCAACTCT TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA

 3481 AATACTGTTC TTCTAGTGTA GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG

 3541 CCTACATACC TCGCTCTGCT AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG

 3601 TGTCTTACCG GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA

 3661 ACGGGGGGTT CGTGCACACA GCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC

 3721 CTACAGCGTG AGCTATGAGA AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT

 3781 CCGGTAAGCG GCAGGGTCGG AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC

 3841 TGGTATCTTT ATAGTCCTGT CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA

 3901 TGCTCGTCAG GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC

 3961 CTGGCCTTTT GCTGGCCTTT TGCTCACATG TTCTTTCCTG CGTTATCCCC TGATTCTGTG

 4021 GATAACCGTA TTACCGCCAT GCATTAGTTA TTAATAGTAA TCAATTACGG GGTCATTAGT

 4081 TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG

 4141 ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC

 4201 AATAGGGACT TTCCATTGAC GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC

 4261 AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT GACGTCAATG ACGGTAAATG

 4321 GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT

 4381 CTACGTATTA GTCATCGCTA TTACCATGGT GATGCGGTTT TGGCAGTACA TCAATGGGCG

 4441 TGGATAGCGG TTTGACTCAC GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG

 4501 TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT CGTAACAACT CCGCCCCATT

 4561 GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTGGTTTAGT

 4621 GAACCGTCAG ATCCGCTAGC TAAGGCCTCT GTCGA

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