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        pEGFP-N2质粒图谱及信息

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        pEGFP-N2质粒图谱及信息

        pEGFP-N2质粒图谱及信息

        Restriction Map and Multiple Cloning Site of pEGFP-N2. (Unique restriction sites are in color or bold.) The Not I site follows the EGFP stop codon. The Nhe I site cannot be used for fusions since it contains an in-frame stop codon. The Xba I site ~undefined) is methylated in the DNA provided by CLONTECH. If you wish to digest the vector with this enzyme, you will need to transform the vector into a dam- host and make fresh DNA.

         

        Description

        pEGFP-N2 encodes a red-shifted variant of wild-type GFP (1-3) which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.) pEGFP-N2 encodes the GFPmut1 variant (4) which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. The coding sequence of the EGFP gene contains more than 190 silent base changes which correspond to human codon-usage preferences (5). Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site (6) to further increase the translation efficiency in eukaryotic cells. The MCS in pEGFP-N2 is between the immediate early promoter of CMV (PCMV IE ) and the EGFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of EGFP if they are in the same reading frame as EGFP and there are no intervening stop codons. SV40 polyadenylation signals downstream of the EGFP gene direct proper processing of the 3' end of the EGFP 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 thymidine kinase gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette (Pamp ) expresses kanamycin resistance in E. coli . The pGFP-C1 backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.


        Use

        Fusions to the N-terminus of EGFP retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo . The target gene should be cloned into pEGFP-N2 so that it is in frame with the EGFP coding sequences, with no intervening in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant EGFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (7). pEGFP-N2 can also be used simply to express EGFP in a cell line of interest (e.g., as a transfection marker).


        Feature Locations

      • Human cytomegalovirus (CMV) immediate early promoter: 1-589
          Enhancer region: 59-465
          TATA box: 554-560
          Transcription start point: 583
          C-> G mutation to remove Sac I site: 569
      • MCS: 591-665
      • Enhanced green fluorescent protein gene
          Kozak consensus translation initiation site: 676-686
          Start codon (ATG): 683-685; Stop codon: 1400-1402
          Insertion of Val at position 2: 686-688
          GFPmut1 chromophore mutations (Phe-64 to Leu; Ser-65 to Thr): 875-880
          His-231 to Leu mutation: 1377
      • SV40 early mRNA polyadenylation signal
          Polyadenylation signals: 1556-1561 & 1585-1590
          mRNA 3' ends: 1594 & 1606
      • f1 single-strand DNA origin: 1653-2108
        (packages the noncoding strand of EGFP)
      • Ampicillin resistance (beta-lactamase) promoter
          -35 region: 2170-2175; -10 region: 2193-2198
          Transcription start point: 2205
      • SV40 origin of replication: 2449-2584
      • SV40 early promoter
          Enhancer (72-bp tandem repeats): 2282-2353 & 2354-2425
          21-bp repeats: 2429-2449, 2450-2470, & 2472-2492
          Early promoter element: 2505-2511
          Major transcription start points: 2501, 2539, 2545, & 2550
      • Kanamycin/neomycin resistance gene
          Neomycin phosphotransferase coding sequences:
          Start codon (ATG): 2633-2635; stop codon: 3425-3427
          G-> A mutation to remove Pst I site: 2815
          C-> A (Arg to Ser) mutation to remove Bss H II site: 3161
      • Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
        Polyadenylation signals: 3663-3668 & 3676-3681
      • pUC plasmid replication origin: 4012-4655

      • Primer Sites

      • EGFP-N Sequencing Primer (#6479-1): 749-728
      • EGFP-C Sequencing Primer (#6478-1): 1336-1357
      • 全序列:

        TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG
        CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT
        GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA
        ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC
        AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA
        CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC
        CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG
        ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG
        GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT
        ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTA
        CCGGACTCAG ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGG
        GATCCACCGG CCGGTCGCCA CCATGGTGAG CAAGGGCGAG GAGCTGTTCA CCGGGGTGGT
        GCCCATCCTG GTCGAGCTGG ACGGCGACGT AAACGGCCAC AAGTTCAGCG TGTCCGGCGA
        GGGCGAGGGC GATGCCACCT ACGGCAAGCT GACCCTGAAG TTCATCTGCA CCACCGGCAA
        GCTGCCCGTG CCCTGGCCCA CCCTCGTGAC CACCCTGACC TACGGCGTGC AGTGCTTCAG
        CCGCTACCCC GACCACATGA AGCAGCACGA CTTCTTCAAG TCCGCCATGC CCGAAGGCTA
        CGTCCAGGAG CGCACCATCT TCTTCAAGGA CGACGGCAAC TACAAGACCC GCGCCGAGGT
        GAAGTTCGAG GGCGACACCC TGGTGAACCG CATCGAGCTG AAGGGCATCG ACTTCAAGGA
        GGACGGCAAC ATCCTGGGGC ACAAGCTGGA GTACAACTAC AACAGCCACA ACGTCTATAT
        CATGGCCGAC AAGCAGAAGA ACGGCATCAA GGTGAACTTC AAGATCCGCC ACAACATCGA
        GGACGGCAGC GTGCAGCTCG CCGACCACTA CCAGCAGAAC ACCCCCATCG GCGACGGCCC
        CGTGCTGCTG CCCGACAACC ACTACCTGAG CACCCAGTCC GCCCTGAGCA AAGACCCCAA
        CGAGAAGCGC GATCACATGG TCCTGCTGGA GTTCGTGACC GCCGCCGGGA TCACTCTCGG
        CATGGACGAG CTGTACAAGT AAAGCGGCCG CGACTCTAGA TCATAATCAG CCATACCACA
        TTTGTAGAGG TTTTACTTGC TTTAAAAAAC CTCCCACACC TCCCCCTGAA CCTGAAACAT
        AAAATGAATG CAATTGTTGT TGTTAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA
        AGCAATAGCA TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC TAGTTGTGGT
        TTGTCCAAAC TCATCAATGT ATCTTAAGGC GTAAATTGTA AGCGTTAATA TTTTGTTAAA
        ATTCGCGTTA AATTTTTGTT AAATCAGCTC ATTTTTTAAC CAATAGGCCG AAATCGGCAA
        AATCCCTTAT AAATCAAAAG AATAGACCGA GATAGGGTTG AGTGTTGTTC CAGTTTGGAA
        CAAGAGTCCA CTATTAAAGA ACGTGGACTC CAACGTCAAA GGGCGAAAAA CCGTCTATCA
        GGGCGATGGC CCACTACGTG AACCATCACC CTAATCAAGT TTTTTGGGGT CGAGGTGCCG
        TAAAGCACTA AATCGGAACC CTAAAGGGAG CCCCCGATTT AGAGCTTGAC GGGGAAAGCC
        GGCGAACGTG GCGAGAAAGG AAGGGAAGAA AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC
        AAGTGTAGCG GTCACGCTGC GCGTAACCAC CACACCCGCC GCGCTTAATG CGCCGCTACA
        GGGCGCGTCA GGTGGCACTT TTCGGGGAAA TGTGCGCGGA ACCCCTATTT GTTTATTTTT
        CTAAATACAT TCAAATATGT ATCCGCTCAT GAGACAATAA CCCTGATAAA TGCTTCAATA
        ATATTGAAAA AGGAAGAGTC CTGAGGCGGA AAGAACCAGC TGTGGAATGT GTGTCAGTTA
        GGGTGTGGAA AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT
        TAGTCAGCAA CCAGGTGTGG AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC
        ATGCATCTCA ATTAGTCAGC AACCATAGTC CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA
        ACTCCGCCCA GTTCCGCCCA TTCTCCGCCC CATGGCTGAC TAATTTTTTT TATTTATGCA
        GAGGCCGAGG CCGCCTCGGC CTCTGAGCTA TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA
        GGCCTAGGCT TTTGCAAAGA TCGATCAAGA GACAGGATGA GGATCGTTTC GCATGATTGA
        ACAAGATGGA TTGCACGCAG GTTCTCCGGC CGCTTGGGTG GAGAGGCTAT TCGGCTATGA
        CTGGGCACAA CAGACAATCG GCTGCTCTGA TGCCGCCGTG TTCCGGCTGT CAGCGCAGGG
        GCGCCCGGTT CTTTTTGTCA AGACCGACCT GTCCGGTGCC CTGAATGAAC TGCAAGACGA
        GGCAGCGCGG CTATCGTGGC TGGCCACGAC GGGCGTTCCT TGCGCAGCTG TGCTCGACGT
        TGTCACTGAA GCGGGAAGGG ACTGGCTGCT ATTGGGCGAA GTGCCGGGGC AGGATCTCCT
        GTCATCTCAC CTTGCTCCTG CCGAGAAAGT ATCCATCATG GCTGATGCAA TGCGGCGGCT
        GCATACGCTT GATCCGGCTA CCTGCCCATT CGACCACCAA GCGAAACATC GCATCGAGCG
        AGCACGTACT CGGATGGAAG CCGGTCTTGT CGATCAGGAT GATCTGGACG AAGAGCATCA
        GGGGCTCGCG CCAGCCGAAC TGTTCGCCAG GCTCAAGGCG AGCATGCCCG ACGGCGAGGA
        TCTCGTCGTG ACCCATGGCG ATGCCTGCTT GCCGAATATC ATGGTGGAAA ATGGCCGCTT
        TTCTGGATTC ATCGACTGTG GCCGGCTGGG TGTGGCGGAC CGCTATCAGG ACATAGCGTT
        GGCTACCCGT GATATTGCTG AAGAGCTTGG CGGCGAATGG GCTGACCGCT TCCTCGTGCT
        TTACGGTATC GCCGCTCCCG ATTCGCAGCG CATCGCCTTC TATCGCCTTC TTGACGAGTT
        CTTCTGAGCG GGACTCTGGG GTTCGAAATG ACCGACCAAG CGACGCCCAA CCTGCCATCA
        CGAGATTTCG ATTCCACCGC CGCCTTCTAT GAAAGGTTGG GCTTCGGAAT CGTTTTCCGG
        GACGCCGGCT GGATGATCCT CCAGCGCGGG GATCTCATGC TGGAGTTCTT CGCCCACCCT
        AGGGGGAGGC TAACTGAAAC ACGGAAGGAG ACAATACCGG AAGGAACCCG CGCTATGACG
        GCAATAAAAA GACAGAATAA AACGCACGGT GTTGGGTCGT TTGTTCATAA ACGCGGGGTT
        CGGTCCCAGG GCTGGCACTC TGTCGATACC CCACCGAGAC CCCATTGGGG CCAATACGCC
        CGCGTTTCTT CCTTTTCCCC ACCCCACCCC CCAAGTTCGG GTGAAGGCCC AGGGCTCGCA
        GCCAACGTCG GGGCGGCAGG CCCTGCCATA GCCTCAGGTT ACTCATATAT ACTTTAGATT
        GATTTAAAAC TTCATTTTTA ATTTAAAAGG ATCTAGGTGA AGATCCTTTT TGATAATCTC
        ATGACCAAAA TCCCTTAACG TGAGTTTTCG TTCCACTGAG CGTCAGACCC CGTAGAAAAG
        ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT CTGCGCGTAA TCTGCTGCTT GCAAACAAAA
        AAACCACCGC TACCAGCGGT GGTTTGTTTG CCGGATCAAG AGCTACCAAC TCTTTTTCCG
        AAGGTAACTG GCTTCAGCAG AGCGCAGATA CCAAATACTG TCCTTCTAGT GTAGCCGTAG
        TTAGGCCACC ACTTCAAGAA CTCTGTAGCA CCGCCTACAT ACCTCGCTCT GCTAATCCTG
        TTACCAGTGG CTGCTGCCAG TGGCGATAAG TCGTGTCTTA CCGGGTTGGA CTCAAGACGA
        TAGTTACCGG ATAAGGCGCA GCGGTCGGGC TGAACGGGGG GTTCGTGCAC ACAGCCCAGC
        TTGGAGCGAA CGACCTACAC CGAACTGAGA TACCTACAGC GTGAGCTATG AGAAAGCGCC
        ACGCTTCCCG AAGGGAGAAA GGCGGACAGG TATCCGGTAA GCGGCAGGGT CGGAACAGGA
        GAGCGCACGA GGGAGCTTCC AGGGGGAAAC GCCTGGTATC TTTATAGTCC TGTCGGGTTT
        CGCCACCTCT GACTTGAGCG TCGATTTTTG TGATGCTCGT CAGGGGGGCG GAGCCTATGG
        AAAAACGCCA GCAACGCGGC CTTTTTACGG TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC
        ATGTTCTTTC CTGCGTTATC CCCTGATTCT GTGGATAACC GTATTACCGC CATGCAT

        Sequence (text)
        Sequence (pdf)
        Restriction Digest (pdf)
        Description
        Use
        Feature Locations
        Primer Sites

         

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