pmCherry-1

pmCherry-1

pmCherry-1载体基本信息：

pmCherry-1载体质粒图谱和多克隆位点信息：

pmCherry-1载体描述：

pmCherry-1 is a reporter vector that allows the functional analysis of promoters and promoter/enhancer combinations cloned into its multiple cloning site (MCS). The vector encodes mCherry, a mutant fluorescent protein derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed (excitation and emission maxima: 587 nm and 610 nm, respectively; 1). The mCherry coding sequence has been human codon-optimized to allow optimal expression after the insertion of a functional promoter into the MCS (2).

SV40 polyadenylation signals downstream of the mCherry gene direct proper processing of the 3' end of the mCherry mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 large 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 (PSV40 e), the

neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter (PKanr) upstream of the cassette allows kanamycin resistance in E. coli.

pmCherry can be used as an in vivo reporter of gene expression. Promoters should be cloned into the pmCherry-1 MCS upstream from the mCherry coding sequence. Without the addition of a functionalpromoter, this vector will not express mCherry. The mCherry vector can be transfected into mammalian cells using any standard method. If required, stable transfectants can be selected using G418 (3).

For Western analysis, either the Living Colors DsRed Polyclonal Antibody (Cat. No. 632496) or the DsRed Monoclonal Antibody (Cat. Nos. 632392 and 632393) can be used to detect the mCherry protein.

pmCherry-1载体特征：

MCS (multiple cloning site): 12–83

 mCherry (human codon optimized)

Kozak consensus translation initiation site: 90–100

Start codon (ATG): 97–99; Stop codon: 805-807

 SV40 early polyA+ signals

Polyadenylation signals: 959–964 & 988–993; mRNA 3' ends: 997 & 1009

 f1 origin of replication: 1056–1511 (complementary)

 PKanr (bacterial promoter for Kanr gene expression)

–35 region: 1573–1578; –10 region: 1596–1601

Transcription start point: 1608

 PSV40 e (SV40 early promoter and enhancer sequences)

Enhancer (72-bp tandem repeats): 1683–1756 & 1757–1828

21-bp repeats: 1832–1852, 1853–1873 & 1875–1895

Early promoter element: 1918–1924

Major transcription start points: 1904, 1942, 1948 & 1953

 SV40 origin of replication: 1852–1987

 Kanamycin/neomycin resistance geneNeomycin phosphotransferase coding sequences:

Start codon (ATG): 2036–2038; stop codon: 2828–2830

G→A mutation to remove PstI site: 2218

C→A (Arg→Ser) mutation to remove BssHII site: 2564

 HSV TK polyA+ (herpes simplex virus thymidine kinase polyadenylation signals): 3066–3071 & 3079–3084

 pUC plasmid replication origin: 3415–4058

质粒的扩增：

Suitable host strains: DH5慣, 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 關g/ml) in E. coli hosts.

 E. coli replication origin: pUC

 Copy number: ~500

Excitation and emission maxima of mCherry

 Excitation maximum = 587 nm

 Emission maximum = 610 nm

pmCherry-1载体序列：

ORIGIN

    1 TAGTTATTAC TAGCGCTACC GGACTCAGAT CTCGAGCTCA AGCTTCGAAT TCTGCAGTCG

   61 ACGGTACCGC GGGCCCGGGA TCCACCGGTC GCCACCATGG TGAGCAAGGG CGAGGAGGAT

  121 AACATGGCCA TCATCAAGGA GTTCATGCGC TTCAAGGTGC ACATGGAGGG CTCCGTGAAC

  181 GGCCACGAGT TCGAGATCGA GGGCGAGGGC GAGGGCCGCC CCTACGAGGG CACCCAGACC

  241 GCCAAGCTGA AGGTGACCAA GGGTGGCCCC CTGCCCTTCG CCTGGGACAT CCTGTCCCCT

  301 CAGTTCATGT ACGGCTCCAA GGCCTACGTG AAGCACCCCG CCGACATCCC CGACTACTTG

  361 AAGCTGTCCT TCCCCGAGGG CTTCAAGTGG GAGCGCGTGA TGAACTTCGA GGACGGCGGC

  421 GTGGTGACCG TGACCCAGGA CTCCTCCCTG CAGGACGGCG AGTTCATCTA CAAGGTGAAG

  481 CTGCGCGGCA CCAACTTCCC CTCCGACGGC CCCGTAATGC AGAAGAAGAC CATGGGCTGG

  541 GAGGCCTCCT CCGAGCGGAT GTACCCCGAG GACGGCGCCC TGAAGGGCGA GATCAAGCAG

  601 AGGCTGAAGC TGAAGGACGG CGGCCACTAC GACGCTGAGG TCAAGACCAC CTACAAGGCC

  661 AAGAAGCCCG TGCAGCTGCC CGGCGCCTAC AACGTCAACA TCAAGTTGGA CATCACCTCC

  721 CACAACGAGG ACTACACCAT CGTGGAACAG TACGAACGCG CCGAGGGCCG CCACTCCACC

  781 GGCGGCATGG ACGAGCTGTA CAAGTAGCGG CCGCGACTCT AGATCATAAT CAGCCATACC

  841 ACATTTGTAG AGGTTTTACT TGCTTTAAAA AACCTCCCAC ACCTCCCCCT GAACCTGAAA

  901 CATAAAATGA ATGCAATTGT TGTTGTTAAC TTGTTTATTG CAGCTTATAA TGGTTACAAA

  961 TAAAGCAATA GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCA TTCTAGTTGT

 1021 GGTTTGTCCA AACTCATCAA TGTATCTTAA GGCGTAAATT GTAAGCGTTA ATATTTTGTT

 1081 AAAATTCGCG TTAAATTTTT GTTAAATCAG CTCATTTTTT AACCAATAGG CCGAAATCGG

 1141 CAAAATCCCT TATAAATCAA AAGAATAGAC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG

 1201 GAACAAGAGT CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA

 1261 TCAGGGCGAT GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG

 1321 CCGTAAAGCA CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA

 1381 GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT

 1441 GGCAAGTGTA GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT

 1501 ACAGGGCGCG TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT

 1561 TTTCTAAATA CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA

 1621 ATAATATTGA AAAAGGAAGA GTCCTGAGGC GGAAAGAACC AGCTGTGGAA TGTGTGTCAG

 1681 TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA GTATGCAAAG CATGCATCTC

 1741 AATTAGTCAG CAACCAGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA

 1801 AGCATGCATC TCAATTAGTC AGCAACCATA GTCCCGCCCC TAACTCCGCC CATCCCGCCC

 1861 CTAACTCCGC CCAGTTCCGC CCATTCTCCG CCCCATGGCT GACTAATTTT TTTTATTTAT

 1921 GCAGAGGCCG AGGCCGCCTC GGCCTCTGAG CTATTCCAGA AGTAGTGAGG AGGCTTTTTT

 1981 GGAGGCCTAG GCTTTTGCAA AGATCGATCA AGAGACAGGA TGAGGATCGT TTCGCATGAT

 2041 TGAACAAGAT GGATTGCACG CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC TATTCGGCTA

 2101 TGACTGGGCA CAACAGACAA TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA

 2161 GGGGCGCCCG GTTCTTTTTG TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAAGA

 2221 CGAGGCAGCG CGGCTATCGT GGCTGGCCAC GACGGGCGTT CCTTGCGCAG CTGTGCTCGA

 2281 CGTTGTCACT GAAGCGGGAA GGGACTGGCT GCTATTGGGC GAAGTGCCGG GGCAGGATCT

 2341 CCTGTCATCT CACCTTGCTC CTGCCGAGAA AGTATCCATC ATGGCTGATG CAATGCGGCG

 2401 GCTGCATACG CTTGATCCGG CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA

 2461 GCGAGCACGT ACTCGGATGG AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA

 2521 TCAGGGGCTC GCGCCAGCCG AACTGTTCGC CAGGCTCAAG GCGAGCATGC CCGACGGCGA

 2581 GGATCTCGTC GTGACCCATG GCGATGCCTG CTTGCCGAAT ATCATGGTGG AAAATGGCCG

 2641 CTTTTCTGGA TTCATCGACT GTGGCCGGCT GGGTGTGGCG GACCGCTATC AGGACATAGC

 2701 GTTGGCTACC CGTGATATTG CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT

 2761 GCTTTACGGT ATCGCCGCTC CCGATTCGCA GCGCATCGCC TTCTATCGCC TTCTTGACGA

 2821 GTTCTTCTGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC CAACCTGCCA

 2881 TCACGAGATT TCGATTCCAC CGCCGCCTTC TATGAAAGGT TGGGCTTCGG AATCGTTTTC

 2941 CGGGACGCCG GCTGGATGAT CCTCCAGCGC GGGGATCTCA TGCTGGAGTT CTTCGCCCAC

 3001 CCTAGGGGGA GGCTAACTGA AACACGGAAG GAGACAATAC CGGAAGGAAC CCGCGCTATG

 3061 ACGGCAATAA AAAGACAGAA TAAAACGCAC GGTGTTGGGT CGTTTGTTCA TAAACGCGGG

 3121 GTTCGGTCCC AGGGCTGGCA CTCTGTCGAT ACCCCACCGA GACCCCATTG GGGCCAATAC

 3181 GCCCGCGTTT CTTCCTTTTC CCCACCCCAC CCCCCAAGTT CGGGTGAAGG CCCAGGGCTC

 3241 GCAGCCAACG TCGGGGCGGC AGGCCCTGCC ATAGCCTCAG GTTACTCATA TATACTTTAG

 3301 ATTGATTTAA AACTTCATTT TTAATTTAAA AGGATCTAGG TGAAGATCCT TTTTGATAAT

 3361 CTCATGACCA AAATCCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAA

 3421 AAGATCAAAG GATCTTCTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTGCAAACA

 3481 AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC AAGAGCTACC AACTCTTTTT

 3541 CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG ATACCAAATA CTGTTCTTCT AGTGTAGCCG

 3601 TAGTTAGGCC ACCACTTCAA GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGCTAATC

 3661 CTGTTACCAG TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGA

 3721 CGATAGTTAC CGGATAAGGC GCAGCGGTCG GGCTGAACGG GGGGTTCGTG CACACAGCCC

 3781 AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC AGCGTGAGCT ATGAGAAAGC

 3841 GCCACGCTTC CCGAAGGGAG AAAGGCGGAC AGGTATCCGG TAAGCGGCAG GGTCGGAACA

 3901 GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGTCGGG

 3961 TTTCGCCACC TCTGACTTGA GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA

 4021 TGGAAAAACG CCAGCAACGC GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT

 4081 CACATGTTCT TTCCTGCGTT ATCCCCTGAT TCTGTGGATA ACCGTATTAC CGCCATGCAT

//

pmCherry-1载体其他相关的其他类型载体：