pIRES2-DsRed2

pIRES2-DsRed2载体基本信息

pIRES2-DsRed2载体质粒图谱和多克隆位点信息

pIRES2-DsRed2载体简介

pIRES2-DsRed2 contains the internal ribosome entry site (IRES; 1, 2) of the encephalomyocarditis virus (ECMV) between the MCS and the Discosoma sp. red fluorescent protein (DsRed2; 3, 4) coding region. This permits both the gene of interest (cloned into the MCS) and the DsRed2 gene to be translated from a single bicistronic mRNA. pIRES2-DsRed2 is designed for the efficient selection (by flow cytometry or other methods) of transiently transfected mammalian cells expressing DsRed2 and the protein of interest. Thisvector can express DsRed2 alone at lower signal intensity. The vector can also be used to obtain stably transfected cell lines by drug and clonal selection. DsRed2 is a human codon-optimized (5) DsRed variant engineered for faster maturation and lower non-specific aggregation. The MCS in pIRES2-DsRed2 is located between the immediate early promoter of cytomegalovirus (PCMV IE) and the IRES sequence. SV40 polyadenylation signals downstream of the DsRed2 gene direct proper processing of the 3' end of the bicistronic mRNA.

The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. 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. The pIRES2-DsRed2 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

pIRES2-DsRed2 can be used to quickly identify cells expressing a gene of interest by screening for DsRed2 fluorescence. Genes inserted into the MCS should include the initiating ATG codon. Selection of DsRed2-positive cells is possible 24 hr after transfection by flow cytometry or fluorescence microscopy. However, in some cases, up to 48 hr may be required for detection of red-emitting cells. pIRES2-DsRed2 and its derivatives can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (6).

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 JM101 or XL1-Blue.

• 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

pIRES2-DsRed2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 GATCCGCCCC TCTCCCTCCC CCCCCCCTAA CGTTACTGGC CGAAGCCGCT TGGAATAAGG

  721 CCGGTGTGCG TTTGTCTATA TGTTATTTTC CACCATATTG CCGTCTTTTG GCAATGTGAG

  781 GGCCCGGAAA CCTGGCCCTG TCTTCTTGAC GAGCATTCCT AGGGGTCTTT CCCCTCTCGC

  841 CAAAGGAATG CAAGGTCTGT TGAATGTCGT GAAGGAAGCA GTTCCTCTGG AAGCTTCTTG

  901 AAGACAAACA ACGTCTGTAG CGACCCTTTG CAGGCAGCGG AACCCCCCAC CTGGCGACAG

  961 GTGCCTCTGC GGCCAAAAGC CACGTGTATA AGATACACCT GCAAAGGCGG CACAACCCCA

 1021 GTGCCACGTT GTGAGTTGGA TAGTTGTGGA AAGAGTCAAA TGGCTCTCCT CAAGCGTATT

 1081 CAACAAGGGG CTGAAGGATG CCCAGAAGGT ACCCCATTGT ATGGGATCTG ATCTGGGGCC

 1141 TCGGTGCACA TGCTTTACAT GTGTTTAGTC GAGGTTAAAA AAACGTCTAG GCCCCCCGAA

 1201 CCACGGGGAC GTGGTTTTCC TTTGAAAAAC ACGATGATAA TATGGCCACA ACCATGGCCT

 1261 CCTCCGAGAA CGTCATCACC GAGTTCATGC GCTTCAAGGT GCGCATGGAG GGCACCGTGA

 1321 ACGGCCACGA GTTCGAGATC GAGGGCGAGG GCGAGGGCCG CCCCTACGAG GGCCACAACA

 1381 CCGTGAAGCT GAAGGTGACC AAGGGCGGCC CCCTGCCCTT CGCCTGGGAC ATCCTGTCCC

 1441 CCCAGTTCCA GTACGGCTCC AAGGTGTACG TGAAGCACCC CGCCGACATC CCCGACTACA

 1501 AGAAGCTGTC CTTCCCCGAG GGCTTCAAGT GGGAGCGCGT GATGAACTTC GAGGACGGCG

 1561 GCGTGGCGAC CGTGACCCAG GACTCCTCCC TGCAGGACGG CTGCTTCATC TACAAGGTGA

 1621 AGTTCATCGG CGTGAACTTC CCCTCCGACG GCCCCGTGAT GCAGAAGAAG ACCATGGGCT

 1681 GGGAGGCCTC CACCGAGCGC CTGTACCCCC GCGACGGCGT GCTGAAGGGC GAGACCCACA

 1741 AGGCCCTGAA GCTGAAGGAC GGCGGCCACT ACCTGGTGGA GTTCAAGTCT ATCTACATGG

 1801 CCAAGAAGCC CGTGCAGCTG CCCGGCTACT ACTACGTGGA CGCCAAGCTG GACATCACCT

 1861 CCCACAACGA GGACTACACC ATCGTGGAGC AGTACGAGCG CACCGAGGGC CGCCACCACC

 1921 TGTTCCTGTA GCGGCCGCGA CTCTAGATCA TAATCAGCCA TACCACATTT GTAGAGGTTT

 1981 TACTTGCTTT AAAAAACCTC CCACACCTCC CCCTGAACCT GAAACATAAA ATGAATGCAA

 2041 TTGTTGTTGT TAACTTGTTT ATTGCAGCTT ATAATGGTTA CAAATAAAGC AATAGCATCA

 2101 CAAATTTCAC AAATAAAGCA TTTTTTTCAC TGCATTCTAG TTGTGGTTTG TCCAAACTCA

 2161 TCAATGTATC TTAAGGCGTA AATTGTAAGC GTTAATATTT TGTTAAAATT CGCGTTAAAT

 2221 TTTTGTTAAA TCAGCTCATT TTTTAACCAA TAGGCCGAAA TCGGCAAAAT CCCTTATAAA

 2281 TCAAAAGAAT AGACCGAGAT AGGGTTGAGT GTTGTTCCAG TTTGGAACAA GAGTCCACTA

 2341 TTAAAGAACG TGGACTCCAA CGTCAAAGGG CGAAAAACCG TCTATCAGGG CGATGGCCCA

 2401 CTACGTGAAC CATCACCCTA ATCAAGTTTT TTGGGGTCGA GGTGCCGTAA AGCACTAAAT

 2461 CGGAACCCTA AAGGGAGCCC CCGATTTAGA GCTTGACGGG GAAAGCCGGC GAACGTGGCG

 2521 AGAAAGGAAG GGAAGAAAGC GAAAGGAGCG GGCGCTAGGG CGCTGGCAAG TGTAGCGGTC

 2581 ACGCTGCGCG TAACCACCAC ACCCGCCGCG CTTAATGCGC CGCTACAGGG CGCGTCAGGT

 2641 GGCACTTTTC GGGGAAATGT GCGCGGAACC CCTATTTGTT TATTTTTCTA AATACATTCA

 2701 AATATGTATC CGCTCATGAG ACAATAACCC TGATAAATGC TTCAATAATA TTGAAAAAGG

 2761 AAGAGTCCTG AGGCGGAAAG AACCAGCTGT GGAATGTGTG TCAGTTAGGG TGTGGAAAGT

 2821 CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA

 2881 GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT

 2941 AGTCAGCAAC CATAGTCCCG CCCCTAACTC CGCCCATCCC GCCCCTAACT CCGCCCAGTT

 3001 CCGCCCATTC TCCGCCCCAT GGCTGACTAA TTTTTTTTAT TTATGCAGAG GCCGAGGCCG

 3061 CCTCGGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC CTAGGCTTTT

 3121 GCAAAGATCG ATCAAGAGAC AGGATGAGGA TCGTTTCGCA TGATTGAACA AGATGGATTG

 3181 CACGCAGGTT CTCCGGCCGC TTGGGTGGAG AGGCTATTCG GCTATGACTG GGCACAACAG

 3241 ACAATCGGCT GCTCTGATGC CGCCGTGTTC CGGCTGTCAG CGCAGGGGCG CCCGGTTCTT

 3301 TTTGTCAAGA CCGACCTGTC CGGTGCCCTG AATGAACTGC AAGACGAGGC AGCGCGGCTA

 3361 TCGTGGCTGG CCACGACGGG CGTTCCTTGC GCAGCTGTGC TCGACGTTGT CACTGAAGCG

 3421 GGAAGGGACT GGCTGCTATT GGGCGAAGTG CCGGGGCAGG ATCTCCTGTC ATCTCACCTT

 3481 GCTCCTGCCG AGAAAGTATC CATCATGGCT GATGCAATGC GGCGGCTGCA TACGCTTGAT

 3541 CCGGCTACCT GCCCATTCGA CCACCAAGCG AAACATCGCA TCGAGCGAGC ACGTACTCGG

 3601 ATGGAAGCCG GTCTTGTCGA TCAGGATGAT CTGGACGAAG AGCATCAGGG GCTCGCGCCA

 3661 GCCGAACTGT TCGCCAGGCT CAAGGCGAGC ATGCCCGACG GCGAGGATCT CGTCGTGACC

 3721 CATGGCGATG CCTGCTTGCC GAATATCATG GTGGAAAATG GCCGCTTTTC TGGATTCATC

 3781 GACTGTGGCC GGCTGGGTGT GGCGGACCGC TATCAGGACA TAGCGTTGGC TACCCGTGAT

 3841 ATTGCTGAAG AGCTTGGCGG CGAATGGGCT GACCGCTTCC TCGTGCTTTA CGGTATCGCC

 3901 GCTCCCGATT CGCAGCGCAT CGCCTTCTAT CGCCTTCTTG ACGAGTTCTT CTGAGCGGGA

 3961 CTCTGGGGTT CGAAATGACC GACCAAGCGA CGCCCAACCT GCCATCACGA GATTTCGATT

 4021 CCACCGCCGC CTTCTATGAA AGGTTGGGCT TCGGAATCGT TTTCCGGGAC GCCGGCTGGA

 4081 TGATCCTCCA GCGCGGGGAT CTCATGCTGG AGTTCTTCGC CCACCCTAGG GGGAGGCTAA

 4141 CTGAAACACG GAAGGAGACA ATACCGGAAG GAACCCGCGC TATGACGGCA ATAAAAAGAC

 4201 AGAATAAAAC GCACGGTGTT GGGTCGTTTG TTCATAAACG CGGGGTTCGG TCCCAGGGCT

 4261 GGCACTCTGT CGATACCCCA CCGAGACCCC ATTGGGGCCA ATACGCCCGC GTTTCTTCCT

 4321 TTTCCCCACC CCACCCCCCA AGTTCGGGTG AAGGCCCAGG GCTCGCAGCC AACGTCGGGG

 4381 CGGCAGGCCC TGCCATAGCC TCAGGTTACT CATATATACT TTAGATTGAT TTAAAACTTC

 4441 ATTTTTAATT TAAAAGGATC TAGGTGAAGA TCCTTTTTGA TAATCTCATG ACCAAAATCC

 4501 CTTAACGTGA GTTTTCGTTC CACTGAGCGT CAGACCCCGT AGAAAAGATC AAAGGATCTT

 4561 CTTGAGATCC TTTTTTTCTG CGCGTAATCT GCTGCTTGCA AACAAAAAAA CCACCGCTAC

 4621 CAGCGGTGGT TTGTTTGCCG GATCAAGAGC TACCAACTCT TTTTCCGAAG GTAACTGGCT

 4681 TCAGCAGAGC GCAGATACCA AATACTGTCC TTCTAGTGTA GCCGTAGTTA GGCCACCACT

 4741 TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT AATCCTGTTA CCAGTGGCTG

 4801 CTGCCAGTGG CGATAAGTCG TGTCTTACCG GGTTGGACTC AAGACGATAG TTACCGGATA

 4861 AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT CGTGCACACA GCCCAGCTTG GAGCGAACGA

 4921 CCTACACCGA ACTGAGATAC CTACAGCGTG AGCTATGAGA AAGCGCCACG CTTCCCGAAG

 4981 GGAGAAAGGC GGACAGGTAT CCGGTAAGCG GCAGGGTCGG AACAGGAGAG CGCACGAGGG

 5041 AGCTTCCAGG GGGAAACGCC TGGTATCTTT ATAGTCCTGT CGGGTTTCGC CACCTCTGAC

 5101 TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG GGGGGCGGAG CCTATGGAAA AACGCCAGCA

 5161 ACGCGGCCTT TTTACGGTTC CTGGCCTTTT GCTGGCCTTT TGCTCACATG TTCTTTCCTG

 5221 CGTTATCCCC TGATTCTGTG GATAACCGTA TTACCGCCAT GCAT

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