pDsRed2-1

pDsRed2-1

pDsRed2-1载体基本信息：

pDsRed2-1载体质粒图谱和多克隆位点信息：

pDsRed2-1载体描述：

pDsRed2-1 encodes DsRed2, a DsRed variant engineered for faster maturation and lower non-specific aggregation. Derived from the Discosoma sp. red fluorescent protein (drFP583; 1), DsRed2, like its progenitor DsRed1, contains a series of silent base-pair changes that correspond to human codon-usage preferences for high expression in mammalian cells (2). In addition to these changes, DsRed2 contains six amino acid substitutions: V105A, I161T, and S197A, which result in the more rapid appearance of red fluorescence in transfected cell lines; and R2A, K5E, and K9T, which prevent the protein from aggregating. (DsRed2 may, however, form the same tetrameric structure as DsRed1 [3].) In mammalian cell cultures when DsRed2 is expressed constitutively, red-emitting cells can be detected by fluorescence microscopy within 24 hours of transfection. Large insoluble aggregates of protein, often observed in bacterial and mammalian cell systems expressing DsRed1, are dramatically reduced in cells expressing DsRed2. The faster-maturing, more soluble red fluorescent protein is also well tolerated by host cells; mammalian cell cultures transfected with DsRed2 show no obvious signs of reduced viability—in those cell lines tested, cells expressing DsRed2 display the same morphology (e.g., adherence, light-refraction) and growth characteristics as non-transfected controls.

pDsRed2-1 is a promoterless DsRed2 vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS). Sequences upstream of DsRed2 have been converted to a Kozak consensus translation initiation site (4) to increase translation efficiency in eukaryotic cells. SV40 polyadenylation signals downstream of the DsRed2 gene direct proper processing of the 3' end of the DsRed2 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 expresses kanamycin resistance in E. coli.

DsRed2 can be used as an in vivo reporter of gene expression. Promoters should be cloned into the pDsRed2-1 MCS upstream from the DsRed2 coding sequence. Without addition of a functional promoter, this vector will not express DsRed2. The recombinant DsRed2 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (5).

pDsRed2-1载体含有以下元件：

 MCS: 12–83

 Discosoma sp. human codon-optimized Red Fluorescent Protein (DsRed2) gene

Kozak consensus translation initiation site: 90–100

Start codon (ATG): 97–99; Stop codon: 772–774

 SV40 early mRNA polyadenylation signal

Polyadenylation signals: 926–931 & 955–960

mRNA 3' ends: 964 & 976

 f1 single-strand DNA origin: 1023–1478

(Packages noncoding strand of DsRed2.)

 Ampicillin resistance (b-lactamase) promoter

–35 region: 1540–1545; –10 region: 1563–1568

Transcription start point: 1575

 SV40 origin of replication: 1819–1954

 SV40 early promoter

Enhancer (72-bp tandem repeats): 1650–1723 & 1724–1795

21-bp repeats: 1799–1819, 1820–1840 & 1842–1862

Early promoter element: 1875–1881

Major transcription start points: 1871, 1909, 1915 & 1920

 Kanamycin/neomycin resistance geneNeomycin phosphotransferase coding sequences:

Start codon (ATG): 2003–2005; stop codon: 2795–2797

G→A mutation to remove Pst I site: 2185

C→A (Arg→Ser) mutation to remove BssH II site: 2531

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

Polyadenylation signals: 3033–3038 & 3046–3051

 pUC plasmid replication origin: 3382–4025

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

Red Fluorescent Protein (DsRed2)

 Excitation/Emission Maxima: 558 nm / 583 nm

pDsRed2-1载体序列：

ORIGIN

    1 TAGTTATTAC TAGCGCTACC GGACTCAGAT CTCGAGCTCA AGCTTCGAAT TCTGCAGTCG

   61 ACGGTACCGC GGGCCCGGGA TCCACCGGTC GCCACCATGG CCTCCTCCGA GAACGTCATC

  121 ACCGAGTTCA TGCGCTTCAA GGTGCGCATG GAGGGCACCG TGAACGGCCA CGAGTTCGAG

  181 ATCGAGGGCG AGGGCGAGGG CCGCCCCTAC GAGGGCCACA ACACCGTGAA GCTGAAGGTG

  241 ACCAAGGGCG GCCCCCTGCC CTTCGCCTGG GACATCCTGT CCCCCCAGTT CCAGTACGGC

  301 TCCAAGGTGT ACGTGAAGCA CCCCGCCGAC ATCCCCGACT ACAAGAAGCT GTCCTTCCCC

  361 GAGGGCTTCA AGTGGGAGCG CGTGATGAAC TTCGAGGACG GCGGCGTGGC GACCGTGACC

  421 CAGGACTCCT CCCTGCAGGA CGGCTGCTTC ATCTACAAGG TGAAGTTCAT CGGCGTGAAC

  481 TTCCCCTCCG ACGGCCCCGT GATGCAGAAG AAGACCATGG GCTGGGAGGC CTCCACCGAG

  541 CGCCTGTACC CCCGCGACGG CGTGCTGAAG GGCGAGACCC ACAAGGCCCT GAAGCTGAAG

  601 GACGGCGGCC ACTACCTGGT GGAGTTCAAG TCCATCTACA TGGCCAAGAA GCCCGTGCAG

  661 CTGCCCGGCT ACTACTACGT GGACGCCAAG CTGGACATCA CCTCCCACAA CGAGGACTAC

  721 ACCATCGTGG AGCAGTACGA GCGCACCGAG GGCCGCCACC ACCTGTTCCT GTAGCGGCCG

  781 CGACTCTAGA TCATAATCAG CCATACCACA TTTGTAGAGG TTTTACTTGC TTTAAAAAAC

  841 CTCCCACACC TCCCCCTGAA CCTGAAACAT AAAATGAATG CAATTGTTGT TGTTAACTTG

  901 TTTATTGCAG CTTATAATGG TTACAAATAA AGCAATAGCA TCACAAATTT CACAAATAAA

  961 GCATTTTTTT CACTGCATTC TAGTTGTGGT TTGTCCAAAC TCATCAATGT ATCTTAAGGC

 1021 GTAAATTGTA AGCGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC

 1081 ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG AATAGACCGA

 1141 GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGA ACGTGGACTC

 1201 CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACGTG AACCATCACC

 1261 CTAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAACC CTAAAGGGAG

 1321 CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAAGG AAGGGAAGAA

 1381 AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCTGC GCGTAACCAC

 1441 CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTCA GGTGGCACTT TTCGGGGAAA

 1501 TGTGCGCGGA ACCCCTATTT GTTTATTTTT CTAAATACAT TCAAATATGT ATCCGCTCAT

 1561 GAGACAATAA CCCTGATAAA TGCTTCAATA ATATTGAAAA AGGAAGAGTC CTGAGGCGGA

 1621 AAGAACCAGC TGTGGAATGT GTGTCAGTTA GGGTGTGGAA AGTCCCCAGG CTCCCCAGCA

 1681 GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCAGGTGTGG AAAGTCCCCA

 1741 GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCATAGTC

 1801 CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA GTTCCGCCCA TTCTCCGCCC

 1861 CATGGCTGAC TAATTTTTTT TATTTATGCA GAGGCCGAGG CCGCCTCGGC CTCTGAGCTA

 1921 TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT TTTGCAAAGA TCGATCAAGA

 1981 GACAGGATGA GGATCGTTTC GCATGATTGA ACAAGATGGA TTGCACGCAG GTTCTCCGGC

 2041 CGCTTGGGTG GAGAGGCTAT TCGGCTATGA CTGGGCACAA CAGACAATCG GCTGCTCTGA

 2101 TGCCGCCGTG TTCCGGCTGT CAGCGCAGGG GCGCCCGGTT CTTTTTGTCA AGACCGACCT

 2161 GTCCGGTGCC CTGAATGAAC TGCAAGACGA GGCAGCGCGG CTATCGTGGC TGGCCACGAC

 2221 GGGCGTTCCT TGCGCAGCTG TGCTCGACGT TGTCACTGAA GCGGGAAGGG ACTGGCTGCT

 2281 ATTGGGCGAA GTGCCGGGGC AGGATCTCCT GTCATCTCAC CTTGCTCCTG CCGAGAAAGT

 2341 ATCCATCATG GCTGATGCAA TGCGGCGGCT GCATACGCTT GATCCGGCTA CCTGCCCATT

 2401 CGACCACCAA GCGAAACATC GCATCGAGCG AGCACGTACT CGGATGGAAG CCGGTCTTGT

 2461 CGATCAGGAT GATCTGGACG AAGAGCATCA GGGGCTCGCG CCAGCCGAAC TGTTCGCCAG

 2521 GCTCAAGGCG AGCATGCCCG ACGGCGAGGA TCTCGTCGTG ACCCATGGCG ATGCCTGCTT

 2581 GCCGAATATC ATGGTGGAAA ATGGCCGCTT TTCTGGATTC ATCGACTGTG GCCGGCTGGG

 2641 TGTGGCGGAC CGCTATCAGG ACATAGCGTT GGCTACCCGT GATATTGCTG AAGAGCTTGG

 2701 CGGCGAATGG GCTGACCGCT TCCTCGTGCT TTACGGTATC GCCGCTCCCG ATTCGCAGCG

 2761 CATCGCCTTC TATCGCCTTC TTGACGAGTT CTTCTGAGCG GGACTCTGGG GTTCGAAATG

 2821 ACCGACCAAG CGACGCCCAA CCTGCCATCA CGAGATTTCG ATTCCACCGC CGCCTTCTAT

 2881 GAAAGGTTGG GCTTCGGAAT CGTTTTCCGG GACGCCGGCT GGATGATCCT CCAGCGCGGG

 2941 GATCTCATGC TGGAGTTCTT CGCCCACCCT AGGGGGAGGC TAACTGAAAC ACGGAAGGAG

 3001 ACAATACCGG AAGGAACCCG CGCTATGACG GCAATAAAAA GACAGAATAA AACGCACGGT

 3061 GTTGGGTCGT TTGTTCATAA ACGCGGGGTT CGGTCCCAGG GCTGGCACTC TGTCGATACC

 3121 CCACCGAGAC CCCATTGGGG CCAATACGCC CGCGTTTCTT CCTTTTCCCC ACCCCACCCC

 3181 CCAAGTTCGG GTGAAGGCCC AGGGCTCGCA GCCAACGTCG GGGCGGCAGG CCCTGCCATA

 3241 GCCTCAGGTT ACTCATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG

 3301 ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG

 3361 TTCCACTGAG CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT

 3421 CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT GGTTTGTTTG

 3481 CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG GCTTCAGCAG AGCGCAGATA

 3541 CCAAATACTG TCCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA

 3601 CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG

 3661 TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC

 3721 TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA

 3781 TACCTACAGC GTGAGCTATG AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG

 3841 TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC AGGGGGAAAC

 3901 GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG

 3961 TGATGCTCGT CAGGGGGGCG GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG

 4021 TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCTTTC CTGCGTTATC CCCTGATTCT

 4081 GTGGATAACC GTATTACCGC CATGCAT

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