pAsRed2-N1

pAsRed2-N1载体基本信息

pAsRed2-N1载体质粒图谱和多克隆位点信息

pAsRed2-N1载体简介

pAsRed2-N1 encodes AsRed2, a variant of Anemonia sulcata red fluorescent protein (1, 2). AsRed2 has been engineered for brighter fluorescence (Clontech Laboratories, Inc., unpublished data).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-N1 is positioned between the immediate-early promoter of cytomegalovirus (PCMV IE) and the AsRed2 coding sequence. Thus, genes cloned into the MCS will be expressed as fusions to the N-terminus of AsRed2 if they are in the same reading frame as AsRed2 and there are no intervening stop codons. The SV40 polyadenylation signals (SV40 poly A) downstream of the AsRed2 gene 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. In addition, 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 N 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-N1 so that it is in frame with the AsRed2 coding sequence, with no intervening, in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant pAsRed2-N1 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-N1 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 JM101 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

pAsRed2-N1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 GATCCACCGG TCGCCACCAT GGCCTCTTTG CTGAAGAAGA CCATGCCCTT CAGGACCACC

  721 ATCGAGGGCA CCGTGAACGG CCACTACTTC AAGTGCACCG GCAAGGGCGA GGGCAACCCC

  781 CTGGAGGGCA CCCAGGAGAT GAAGATCGAG GTGATCGAGG GCGGCCCCCT GCCCTTCGCC

  841 TTCCACATCC TGTCCACCTC CTGCATGTAC GGCTCCAAGG CCTTCATCAA GTACGTGTCC

  901 GGCATCCCCG ACTACTTCAA GCAGTCCCTC CCCGAGGGCT TCACCTGGGA GCGCACCACC

  961 ACCTACGAGG ACGGCGGCTT CCTGACCGCC CACCAGGACA CCTCCCTGGA CGGCGACTGC

 1021 CTGGTGTACA AGGTGAAGAT CCTGGGCAAC AACTTCCCCG CCGACGGCCC CGTGATGCAG

 1081 AACAAGGCCG GCCGCTGGGA GCCCTCCACC GAGATCGTGT ACGAGGTGGA CGGCGTGCTG

 1141 CGCGGCCAGT CCCTGATGGC CCTGGAGTGC CCCGGCGGTC GCCACCTGAC CTGCCACCTG

 1201 CACACCACCT ACCGCTCCAA GAAGCCCGCC TCCGCCCTGA AGATGCCCGG CTTCCACTTC

 1261 GAGGACCACC GCATCGAGAT CCTGGAGGAG GTGGAGAAGG GCAAGTGCTA CAAGCAGTAC

 1321 GAGGCCGCCG TGGGCCGCTA CTGCGACGCC GCCCCCTCCA AGCTGGGCCA CAACTGAAGC

 1381 GGCCGCGACT CTAGATCATA ATCAGCCATA CCACATTTGT AGAGGTTTTA CTTGCTTTAA

 1441 AAAACCTCCC ACACCTCCCC CTGAACCTGA AACATAAAAT GAATGCAATT GTTGTTGTTA

 1501 ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA TAGCATCACA AATTTCACAA

 1561 ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC CAAACTCATC AATGTATCTT

 1621 AAGGCGTAAA TTGTAAGCGT TAATATTTTG TTAAAATTCG CGTTAAATTT TTGTTAAATC

 1681 AGCTCATTTT TTAACCAATA GGCCGAAATC GGCAAAATCC CTTATAAATC AAAAGAATAG

 1741 ACCGAGATAG GGTTGAGTGT TGTTCCAGTT TGGAACAAGA GTCCACTATT AAAGAACGTG

 1801 GACTCCAACG TCAAAGGGCG AAAAACCGTC TATCAGGGCG ATGGCCCACT ACGTGAACCA

 1861 TCACCCTAAT CAAGTTTTTT GGGGTCGAGG TGCCGTAAAG CACTAAATCG GAACCCTAAA

 1921 GGGAGCCCCC GATTTAGAGC TTGACGGGGA AAGCCGGCGA ACGTGGCGAG AAAGGAAGGG

 1981 AAGAAAGCGA AAGGAGCGGG CGCTAGGGCG CTGGCAAGTG TAGCGGTCAC GCTGCGCGTA

 2041 ACCACCACAC CCGCCGCGCT TAATGCGCCG CTACAGGGCG CGTCAGGTGG CACTTTTCGG

 2101 GGAAATGTGC GCGGAACCCC TATTTGTTTA TTTTTCTAAA TACATTCAAA TATGTATCCG

 2161 CTCATGAGAC AATAACCCTG ATAAATGCTT CAATAATATT GAAAAAGGAA GAGTCCTGAG

 2221 GCGGAAAGAA CCAGCTGTGG AATGTGTGTC AGTTAGGGTG TGGAAAGTCC CCAGGCTCCC

 2281 CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCAGG TGTGGAAAGT

 2341 CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA

 2401 TAGTCCCGCC CCTAACTCCG CCCATCCCGC CCCTAACTCC GCCCAGTTCC GCCCATTCTC

 2461 CGCCCCATGG CTGACTAATT TTTTTTATTT ATGCAGAGGC CGAGGCCGCC TCGGCCTCTG

 2521 AGCTATTCCA GAAGTAGTGA GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC AAAGATCGAT

 2581 CAAGAGACAG GATGAGGATC GTTTCGCATG ATTGAACAAG ATGGATTGCA CGCAGGTTCT

 2641 CCGGCCGCTT GGGTGGAGAG GCTATTCGGC TATGACTGGG CACAACAGAC AATCGGCTGC

 2701 TCTGATGCCG CCGTGTTCCG GCTGTCAGCG CAGGGGCGCC CGGTTCTTTT TGTCAAGACC

 2761 GACCTGTCCG GTGCCCTGAA TGAACTGCAA GACGAGGCAG CGCGGCTATC GTGGCTGGCC

 2821 ACGACGGGCG TTCCTTGCGC AGCTGTGCTC GACGTTGTCA CTGAAGCGGG AAGGGACTGG

 2881 CTGCTATTGG GCGAAGTGCC GGGGCAGGAT CTCCTGTCAT CTCACCTTGC TCCTGCCGAG

 2941 AAAGTATCCA TCATGGCTGA TGCAATGCGG CGGCTGCATA CGCTTGATCC GGCTACCTGC

 3001 CCATTCGACC ACCAAGCGAA ACATCGCATC GAGCGAGCAC GTACTCGGAT GGAAGCCGGT

 3061 CTTGTCGATC AGGATGATCT GGACGAAGAG CATCAGGGGC TCGCGCCAGC CGAACTGTTC

 3121 GCCAGGCTCA AGGCGAGCAT GCCCGACGGC GAGGATCTCG TCGTGACCCA TGGCGATGCC

 3181 TGCTTGCCGA ATATCATGGT GGAAAATGGC CGCTTTTCTG GATTCATCGA CTGTGGCCGG

 3241 CTGGGTGTGG CGGACCGCTA TCAGGACATA GCGTTGGCTA CCCGTGATAT TGCTGAAGAG

 3301 CTTGGCGGCG AATGGGCTGA CCGCTTCCTC GTGCTTTACG GTATCGCCGC TCCCGATTCG

 3361 CAGCGCATCG CCTTCTATCG CCTTCTTGAC GAGTTCTTCT GAGCGGGACT CTGGGGTTCG

 3421 AAATGACCGA CCAAGCGACG CCCAACCTGC CATCACGAGA TTTCGATTCC ACCGCCGCCT

 3481 TCTATGAAAG GTTGGGCTTC GGAATCGTTT TCCGGGACGC CGGCTGGATG ATCCTCCAGC

 3541 GCGGGGATCT CATGCTGGAG TTCTTCGCCC ACCCTAGGGG GAGGCTAACT GAAACACGGA

 3601 AGGAGACAAT ACCGGAAGGA ACCCGCGCTA TGACGGCAAT AAAAAGACAG AATAAAACGC

 3661 ACGGTGTTGG GTCGTTTGTT CATAAACGCG GGGTTCGGTC CCAGGGCTGG CACTCTGTCG

 3721 ATACCCCACC GAGACCCCAT TGGGGCCAAT ACGCCCGCGT TTCTTCCTTT TCCCCACCCC

 3781 ACCCCCCAAG TTCGGGTGAA GGCCCAGGGC TCGCAGCCAA CGTCGGGGCG GCAGGCCCTG

 3841 CCATAGCCTC AGGTTACTCA TATATACTTT AGATTGATTT AAAACTTCAT TTTTAATTTA

 3901 AAAGGATCTA GGTGAAGATC CTTTTTGATA ATCTCATGAC CAAAATCCCT TAACGTGAGT

 3961 TTTCGTTCCA CTGAGCGTCA GACCCCGTAG AAAAGATCAA AGGATCTTCT TGAGATCCTT

 4021 TTTTTCTGCG CGTAATCTGC TGCTTGCAAA CAAAAAAACC ACCGCTACCA GCGGTGGTTT

 4081 GTTTGCCGGA TCAAGAGCTA CCAACTCTTT TTCCGAAGGT AACTGGCTTC AGCAGAGCGC

 4141 AGATACCAAA TACTGTCCTT CTAGTGTAGC CGTAGTTAGG CCACCACTTC AAGAACTCTG

 4201 TAGCACCGCC TACATACCTC GCTCTGCTAA TCCTGTTACC AGTGGCTGCT GCCAGTGGCG

 4261 ATAAGTCGTG TCTTACCGGG TTGGACTCAA GACGATAGTT ACCGGATAAG GCGCAGCGGT

 4321 CGGGCTGAAC GGGGGGTTCG TGCACACAGC CCAGCTTGGA GCGAACGACC TACACCGAAC

 4381 TGAGATACCT ACAGCGTGAG CTATGAGAAA GCGCCACGCT TCCCGAAGGG AGAAAGGCGG

 4441 ACAGGTATCC GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG CACGAGGGAG CTTCCAGGGG

 4501 GAAACGCCTG GTATCTTTAT AGTCCTGTCG GGTTTCGCCA CCTCTGACTT GAGCGTCGAT

 4561 TTTTGTGATG CTCGTCAGGG GGGCGGAGCC TATGGAAAAA CGCCAGCAAC GCGGCCTTTT

 4621 TACGGTTCCT GGCCTTTTGC TGGCCTTTTG CTCACATGTT CTTTCCTGCG TTATCCCCTG

 4681 ATTCTGTGGA TAACCGTATT ACCGCCATGC AT

//

其他哺乳动物表达载体：