pAsRed2-C1

pAsRed2-C1载体基本信息

pAsRed2-C1载体质粒图谱和多克隆位点信息

pAsRed2-C1载体描述

pAsRed2-C1 encodes AsRed2, a variant of Anemonia sulcata red fluorescent protein (1, 2). AsRed2 has been engineered for brighter fluorescence (Clontech, unpublished data). The AsRed2 coding sequence also contains a series of silent base-pair changes, which correspond to human codon-usage preferences for optimal expression in mammalian cells (3). Additionally, an upstream sequence—located just 5' to the AsRed2 start codon—has been converted to a Kozak consensus translation initiation site (4) to further increase the translation efficiency in eukaryotic cells. The multiple cloning site (MCS) in pAsRed2-C1 is positioned between the AsRed2 coding sequence and a pair of SV40 polyadenylation signals (SV40 poly A). Thus, genes cloned into the MCS will be expressed as fusions to the C-terminus of AsRed2 if they are in the same reading frame as AsRed2 and there are no intervening stop codons. Expression of AsRed2 is driven by the cytomegalovirus immediate-early promoter (PCMV IE). The SV40 poly A signals downstream of the MCS direct proper processing of the 3' end of AsRed2 mRNA.

The vector backbone contains an SV40 origin (SV40 ori) for replication in mammalian cells that express the SV40 T-antigen, a pUC origin of replication (pUC ori) for propagation in E. coli, and an f1 origin (f1 ori) for single-stranded DNA production. A neomycin resistance cassette—consisting of the SV40 early promoter (PSV40e), the neomycin/kanamycin resistance gene of Tn5 (Neor/Kanr), and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK poly A) gene—allows stably transfected eukaryotic cells to be selected using G418 (5). A bacterial promoter (P) upstream of this cassette drives expression of the Neor/Kanr gene in E. coli hosts, which can be selected with kanamycin.

Fusions to the C terminus of AsRed2 retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo (AsRed2 excitation maximum = 576 nm; AsRed2 emission maximum = 592 nm). The target gene should be cloned into pAsRed2-C1 so that it is in frame with the AsRed2 coding sequence, with no intervening, in-frame stop codons. The recombinant pAsRed2-C1 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (available from Clontech; Cat. Nos. 631307 & 631308). We recommend selecting mammalian cell cultures in 500–1,300 μg/ml G418, depending on the cell line. Be sure to establish a kill curve for each cell line and each lot of G418 to determine the optimal selection concentration. Unmodified (i.e., non-recombinant) pAsRed2-C1 can also be used simply to express AsRed2 in cells of interest (e.g., as a transfection marker).

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

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 TTTGCTGAAG AAGACCATGC CCTTCAGGAC CACCATCGAG

  661 GGCACCGTGA ACGGCCACTA CTTCAAGTGC ACCGGCAAGG GCGAGGGCAA CCCCCTGGAG

  721 GGCACCCAGG AGATGAAGAT CGAGGTGATC GAGGGCGGCC CCCTGCCCTT CGCCTTCCAC

  781 ATCCTGTCCA CCTCCTGCAT GTACGGCTCC AAGGCCTTCA TCAAGTACGT GTCCGGCATC

  841 CCCGACTACT TCAAGCAGTC CCTCCCCGAG GGCTTCACCT GGGAGCGCAC CACCACCTAC

  901 GAGGACGGCG GCTTCCTGAC CGCCCACCAG GACACCTCCC TGGACGGCGA CTGCCTGGTG

  961 TACAAGGTGA AGATCCTGGG CAACAACTTC CCCGCCGACG GCCCCGTGAT GCAGAACAAG

 1021 GCCGGCCGCT GGGAGCCCTC CACCGAGATC GTGTACGAGG TGGACGGCGT GCTGCGCGGC

 1081 CAGTCCCTGA TGGCCCTGGA GTGCCCCGGC GGTCGCCACC TGACCTGCCA CCTGCACACC

 1141 ACCTACCGCT CCAAGAAGCC CGCCTCCGCC CTGAAGATGC CCGGCTTCCA CTTCGAGGAC

 1201 CACCGCATCG AGATCCTGGA GGAGGTGGAG AAGGGCAAGT GCTACAAGCA GTACGAGGCC

 1261 GCCGTGGGCC GCTACTGCGA CGCCGCCCCC TCCAAGCTGG GCCACAACTC CGGACTCAGA

 1321 TCTCGAGCTC AAGCTTCGAA TTCTGCAGTC GACGGTACCG CGGGCCCGGG ATCCACCGGA

 1381 TCTAGATAAC TGATCATAAT CAGCCATACC ACATTTGTAG AGGTTTTACT TGCTTTAAAA

 1441 AACCTCCCAC ACCTCCCCCT GAACCTGAAA CATAAAATGA ATGCAATTGT TGTTGTTAAC

 1501 TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA GCATCACAAA TTTCACAAAT

 1561 AAAGCATTTT TTTCACTGCA TTCTAGTTGT GGTTTGTCCA AACTCATCAA TGTATCTTAA

 1621 CGCGTAAATT GTAAGCGTTA ATATTTTGTT AAAATTCGCG TTAAATTTTT GTTAAATCAG

 1681 CTCATTTTTT AACCAATAGG CCGAAATCGG CAAAATCCCT TATAAATCAA AAGAATAGAC

 1741 CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT CCACTATTAA AGAACGTGGA

 1801 CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT GGCCCACTAC GTGAACCATC

 1861 ACCCTAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA CTAAATCGGA ACCCTAAAGG

 1921 GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA

 1981 GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA GCGGTCACGC TGCGCGTAAC

 2041 CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG TCAGGTGGCA CTTTTCGGGG

 2101 AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA CATTCAAATA TGTATCCGCT

 2161 CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA AAAAGGAAGA GTCCTGAGGC

 2221 GGAAAGAACC AGCTGTGGAA TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA

 2281 GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCAGGTG TGGAAAGTCC

 2341 CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCATA

 2401 GTCCCGCCCC TAACTCCGCC CATCCCGCCC CTAACTCCGC CCAGTTCCGC CCATTCTCCG

 2461 CCCCATGGCT GACTAATTTT TTTTATTTAT GCAGAGGCCG AGGCCGCCTC GGCCTCTGAG

 2521 CTATTCCAGA AGTAGTGAGG AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA AGATCGATCA

 2581 AGAGACAGGA TGAGGATCGT TTCGCATGAT TGAACAAGAT GGATTGCACG CAGGTTCTCC

 2641 GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA TCGGCTGCTC

 2701 TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG TCAAGACCGA

 2761 CCTGTCCGGT GCCCTGAATG AACTGCAAGA CGAGGCAGCG CGGCTATCGT GGCTGGCCAC

 2821 GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CGTTGTCACT GAAGCGGGAA GGGACTGGCT

 2881 GCTATTGGGC GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC CTGCCGAGAA

 2941 AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG CTACCTGCCC

 3001 ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT ACTCGGATGG AAGCCGGTCT

 3061 TGTCGATCAG GATGATCTGG ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG AACTGTTCGC

 3121 CAGGCTCAAG GCGAGCATGC CCGACGGCGA GGATCTCGTC GTGACCCATG GCGATGCCTG

 3181 CTTGCCGAAT ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT GTGGCCGGCT

 3241 GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG CTGAAGAGCT

 3301 TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT ATCGCCGCTC CCGATTCGCA

 3361 GCGCATCGCC TTCTATCGCC TTCTTGACGA GTTCTTCTGA GCGGGACTCT GGGGTTCGAA

 3421 ATGACCGACC AAGCGACGCC CAACCTGCCA TCACGAGATT TCGATTCCAC CGCCGCCTTC

 3481 TATGAAAGGT TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT CCTCCAGCGC

 3541 GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCTAGGGGGA GGCTAACTGA AACACGGAAG

 3601 GAGACAATAC CGGAAGGAAC CCGCGCTATG ACGGCAATAA AAAGACAGAA TAAAACGCAC

 3661 GGTGTTGGGT CGTTTGTTCA TAAACGCGGG GTTCGGTCCC AGGGCTGGCA CTCTGTCGAT

 3721 ACCCCACCGA GACCCCATTG GGGCCAATAC GCCCGCGTTT CTTCCTTTTC CCCACCCCAC

 3781 CCCCCAAGTT CGGGTGAAGG CCCAGGGCTC GCAGCCAACG TCGGGGCGGC AGGCCCTGCC

 3841 ATAGCCTCAG GTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT TTAATTTAAA

 3901 AGGATCTAGG TGAAGATCCT TTTTGATAAT CTCATGACCA AAATCCCTTA ACGTGAGTTT

 3961 TCGTTCCACT GAGCGTCAGA CCCCGTAGAA AAGATCAAAG GATCTTCTTG AGATCCTTTT

 4021 TTTCTGCGCG TAATCTGCTG CTTGCAAACA AAAAAACCAC CGCTACCAGC GGTGGTTTGT

 4081 TTGCCGGATC AAGAGCTACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG

 4141 ATACCAAATA CTGTCCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA GAACTCTGTA

 4201 GCACCGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC CAGTGGCGAT

 4261 AAGTCGTGTC TTACCGGGTT GGACTCAAGA CGATAGTTAC CGGATAAGGC GCAGCGGTCG

 4321 GGCTGAACGG GGGGTTCGTG CACACAGCCC AGCTTGGAGC GAACGACCTA CACCGAACTG

 4381 AGATACCTAC AGCGTGAGCT ATGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC

 4441 AGGTATCCGG TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA

 4501 AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA GCGTCGATTT

 4561 TTGTGATGCT CGTCAGGGGG GCGGAGCCTA TGGAAAAACG CCAGCAACGC GGCCTTTTTA

 4621 CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT CACATGTTCT TTCCTGCGTT ATCCCCTGAT

 4681 TCTGTGGATA ACCGTATTAC CGCCATGCAT

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