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RestrictionMapandMultipleCloningSiteofpEGFP-N2.(Uniquerestrictionsitesareincolororbold.)TheNotIsitefollowstheEGFPstopcodon.TheNheIsitecannotbeusedforfusionssinceitcontainsanin-framestopcodon.TheXbaIsite(*)ismethylatedintheDNAprovidedbyCLONTECH.Ifyouwishtodigestthevectorwiththisenzyme,youwillneedtotransformthevectorintoadam-hostandmakefreshDNA. Description pEGFP-N2encodesared-shiftedvariantofwild-typeGFP(1-3)whichhasbeenoptimizedforbrighterfluorescenceandhigherexpressioninmammaliancells.(Excitationmaximum=488nm;emissionmaximum=507nm.)pEGFP-N2encodestheGFPmut1variant(4)whichcontainsthedouble-amino-acidsubstitutionofPhe-64toLeuandSer-65toThr.ThecodingsequenceoftheEGFPgenecontainsmorethan190silentbasechangeswhichcorrespondtohumancodon-usagepreferences(5).SequencesflankingEGFPhavebeenconvertedtoaKozakconsensustranslationinitiationsite(6)tofurtherincreasethetranslationefficiencyineukaryoticcells.TheMCSinpEGFP-N2isbetweentheimmediateearlypromoterofCMV(PCMVIE)andtheEGFPcodingsequences.GenesclonedintotheMCSwillbeexpressedasfusionstotheN-terminusofEGFPiftheyareinthesamereadingframeasEGFPandtherearenointerveningstopcodons.SV40polyadenylationsignalsdownstreamoftheEGFPgenedirectproperprocessingofthe3endoftheEGFPmRNA.ThevectorbackbonealsocontainsanSV40originforreplicationinmammaliancellsexpressingtheSV40T-antigen.Aneomycin-resistancecassette(neor),consistingoftheSV40earlypromoter,theneomycin/kanamycinresistancegeneofTn5,andpolyadenylationsignalsfromtheHerpessimplexthymidinekinasegene,allowsstablytransfectedeukaryoticcellstobeselectedusingG418.Abacterialpromoterupstreamofthiscassette(Pamp)expresseskanamycinresistanceinE.coli.ThepGFP-C1backbonealsoprovidesapUC19originofreplicationforpropagationinE.coliandanf1originforsingle-strandedDNAproduction. Use FusionstotheN-terminusofEGFPretainthefluorescentpropertiesofthenativeproteinallowingthelocalizationofthefusionproteininvivo.ThetargetgeneshouldbeclonedintopEGFP-N2sothatitisinframewiththeEGFPcodingsequences,withnointerveningin-framestopcodons.TheinsertedgeneshouldincludetheinitiatingATGcodon.TherecombinantEGFPvectorcanbetransfectedintomammaliancellsusinganystandardtransfectionmethod.Ifrequired,stabletransformantscanbeselectedusingG418(7).pEGFP-N2canalsobeusedsimplytoexpressEGFPinacelllineofinterest(e.g.,asatransfectionMarker). FeatureLocations PrimerSites 全序列： Sequence(text)Sequence(pdf)RestrictionDigest(pdf)DescriptionUseFeatureLocationsPrimerSites
Enhancerregion:59-465TATAbox:554-560Transcriptionstartpoint:583C->GmutationtoremoveSacIsite:569
Kozakconsensustranslationinitiationsite:676-686Startcodon(ATG):683-685;Stopcodon:1400-1402InsertionofValatposition2:686-688GFPmut1chromophoremutations(Phe-64toLeu;Ser-65toThr):875-880His-231toLeumutation:1377
Polyadenylationsignals:1556-1561&1585-1590mRNA3ends:1594&1606
-35region:2170-2175;-10region:2193-2198Transcriptionstartpoint:2205
Enhancer(72-bptandemrepeats):2282-2353&2354-242521-bprepeats:2429-2449,2450-2470,&2472-2492Earlypromoterelement:2505-2511Majortranscriptionstartpoints:2501,2539,2545,&2550
Neomycinphosphotransferasecodingsequences:Startcodon(ATG):2633-2635;stopcodon:3425-3427G->AmutationtoremovePstIsite:2815C->A(ArgtoSer)mutationtoremoveBssHIIsite:3161
TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCGCGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATTGACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCAATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCCAAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTACATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTACCATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGGATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACGGGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGTACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTACCGGACTCAG ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGGGATCCACCGG CCGGTCGCCA CCATGGTGAG CAAGGGCGAG GAGCTGTTCA CCGGGGTGGTGCCCATCCTG GTCGAGCTGG ACGGCGACGT AAACGGCCAC AAGTTCAGCG TGTCCGGCGAGGGCGAGGGC GATGCCACCT ACGGCAAGCT GACCCTGAAG TTCATCTGCA CCACCGGCAAGCTGCCCGTG CCCTGGCCCA CCCTCGTGAC CACCCTGACC TACGGCGTGC AGTGCTTCAGCCGCTACCCC GACCACATGA AGCAGCACGA CTTCTTCAAG TCCGCCATGC CCGAAGGCTACGTCCAGGAG CGCACCATCT TCTTCAAGGA CGACGGCAAC TACAAGACCC GCGCCGAGGTGAAGTTCGAG GGCGACACCC TGGTGAACCG CATCGAGCTG AAGGGCATCG ACTTCAAGGAGGACGGCAAC ATCCTGGGGC ACAAGCTGGA GTACAACTAC AACAGCCACA ACGTCTATATCATGGCCGAC AAGCAGAAGA ACGGCATCAA GGTGAACTTC AAGATCCGCC ACAACATCGAGGACGGCAGC GTGCAGCTCG CCGACCACTA CCAGCAGAAC ACCCCCATCG GCGACGGCCCCGTGCTGCTG CCCGACAACC ACTACCTGAG CACCCAGTCC GCCCTGAGCA AAGACCCCAACGAGAAGCGC GATCACATGG TCCTGCTGGA GTTCGTGACC GCCGCCGGGA TCACTCTCGGCATGGACGAG CTGTACAAGT AAAGCGGCCG CGACTCTAGA TCATAATCAG CCATACCACATTTGTAGAGG TTTTACTTGC TTTAAAAAAC CTCCCACACC TCCCCCTGAA CCTGAAACATAAAATGAATG CAATTGTTGT TGTTAACTTG TTTATTGCAG CTTATAATGG TTACAAATAAAGCAATAGCA TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC TAGTTGTGGTTTGTCCAAAC TCATCAATGT ATCTTAAGGC GTAAATTGTA AGCGTTAATA TTTTGTTAAAATTCGCGTTA AATTTTTGTT AAATCAGCTC ATTTTTTAAC CAATAGGCCG AAATCGGCAAAATCCCTTAT AAATCAAAAG AATAGACCGA GATAGGGTTG AGTGTTGTTC CAGTTTGGAACAAGAGTCCA CTATTAAAGA ACGTGGACTC CAACGTCAAA GGGCGAAAAA CCGTCTATCAGGGCGATGGC CCACTACGTG AACCATCACC CTAATCAAGT TTTTTGGGGT CGAGGTGCCGTAAAGCACTA AATCGGAACC CTAAAGGGAG CCCCCGATTT AGAGCTTGAC GGGGAAAGCCGGCGAACGTG GCGAGAAAGG AAGGGAAGAA AGCGAAAGGA GCGGGCGCTA GGGCGCTGGCAAGTGTAGCG GTCACGCTGC GCGTAACCAC CACACCCGCC GCGCTTAATG CGCCGCTACAGGGCGCGTCA GGTGGCACTT TTCGGGGAAA TGTGCGCGGA ACCCCTATTT GTTTATTTTTCTAAATACAT TCAAATATGT ATCCGCTCAT GAGACAATAA CCCTGATAAA TGCTTCAATAATATTGAAAA AGGAAGAGTC CTGAGGCGGA AAGAACCAGC TGTGGAATGT GTGTCAGTTAGGGTGTGGAA AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAATTAGTCAGCAA CCAGGTGTGG AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGCATGCATCTCA ATTAGTCAGC AACCATAGTC CCGCCCCTAA CTCCGCCCAT CCCGCCCCTAACTCCGCCCA GTTCCGCCCA TTCTCCGCCC CATGGCTGAC TAATTTTTTT TATTTATGCAGAGGCCGAGG CCGCCTCGGC CTCTGAGCTA TTCCAGAAGT AGTGAGGAGG CTTTTTTGGAGGCCTAGGCT TTTGCAAAGA TCGATCAAGA GACAGGATGA GGATCGTTTC GCATGATTGAACAAGATGGA TTGCACGCAG GTTCTCCGGC CGCTTGGGTG GAGAGGCTAT TCGGCTATGACTGGGCACAA CAGACAATCG GCTGCTCTGA TGCCGCCGTG TTCCGGCTGT CAGCGCAGGGGCGCCCGGTT CTTTTTGTCA AGACCGACCT GTCCGGTGCC CTGAATGAAC TGCAAGACGAGGCAGCGCGG CTATCGTGGC TGGCCACGAC GGGCGTTCCT TGCGCAGCTG TGCTCGACGTTGTCACTGAA GCGGGAAGGG ACTGGCTGCT ATTGGGCGAA GTGCCGGGGC AGGATCTCCTGTCATCTCAC CTTGCTCCTG CCGAGAAAGT ATCCATCATG GCTGATGCAA TGCGGCGGCTGCATACGCTT GATCCGGCTA CCTGCCCATT CGACCACCAA GCGAAACATC GCATCGAGCGAGCACGTACT CGGATGGAAG CCGGTCTTGT CGATCAGGAT GATCTGGACG AAGAGCATCAGGGGCTCGCG CCAGCCGAAC TGTTCGCCAG GCTCAAGGCG AGCATGCCCG ACGGCGAGGATCTCGTCGTG ACCCATGGCG ATGCCTGCTT GCCGAATATC ATGGTGGAAA ATGGCCGCTTTTCTGGATTC ATCGACTGTG GCCGGCTGGG TGTGGCGGAC CGCTATCAGG ACATAGCGTTGGCTACCCGT GATATTGCTG AAGAGCTTGG CGGCGAATGG GCTGACCGCT TCCTCGTGCTTTACGGTATC GCCGCTCCCG ATTCGCAGCG CATCGCCTTC TATCGCCTTC TTGACGAGTTCTTCTGAGCG GGACTCTGGG GTTCGAAATG ACCGACCAAG CGACGCCCAA CCTGCCATCACGAGATTTCG ATTCCACCGC CGCCTTCTAT GAAAGGTTGG GCTTCGGAAT CGTTTTCCGGGACGCCGGCT GGATGATCCT CCAGCGCGGG GATCTCATGC TGGAGTTCTT CGCCCACCCTAGGGGGAGGC TAACTGAAAC ACGGAAGGAG ACAATACCGG AAGGAACCCG CGCTATGACGGCAATAAAAA GACAGAATAA AACGCACGGT GTTGGGTCGT TTGTTCATAA ACGCGGGGTTCGGTCCCAGG GCTGGCACTC TGTCGATACC CCACCGAGAC CCCATTGGGG CCAATACGCCCGCGTTTCTT CCTTTTCCCC ACCCCACCCC CCAAGTTCGG GTGAAGGCCC AGGGCTCGCAGCCAACGTCG GGGCGGCAGG CCCTGCCATA GCCTCAGGTT ACTCATATAT ACTTTAGATTGATTTAAAAC TTCATTTTTA ATTTAAAAGG ATCTAGGTGA AGATCCTTTT TGATAATCTCATGACCAAAA TCCCTTAACG TGAGTTTTCG TTCCACTGAG CGTCAGACCC CGTAGAAAAGATCAAAGGAT CTTCTTGAGA TCCTTTTTTT CTGCGCGTAA TCTGCTGCTT GCAAACAAAAAAACCACCGC TACCAGCGGT GGTTTGTTTG CCGGATCAAG AGCTACCAAC TCTTTTTCCGAAGGTAACTG GCTTCAGCAG AGCGCAGATA CCAAATACTG TCCTTCTAGT GTAGCCGTAGTTAGGCCACC ACTTCAAGAA CTCTGTAGCA CCGCCTACAT ACCTCGCTCT GCTAATCCTGTTACCAGTGG CTGCTGCCAG TGGCGATAAG TCGTGTCTTA CCGGGTTGGA CTCAAGACGATAGTTACCGG ATAAGGCGCA GCGGTCGGGC TGAACGGGGG GTTCGTGCAC ACAGCCCAGCTTGGAGCGAA CGACCTACAC CGAACTGAGA TACCTACAGC GTGAGCTATG AGAAAGCGCCACGCTTCCCG AAGGGAGAAA GGCGGACAGG TATCCGGTAA GCGGCAGGGT CGGAACAGGAGAGCGCACGA GGGAGCTTCC AGGGGGAAAC GCCTGGTATC TTTATAGTCC TGTCGGGTTTCGCCACCTCT GACTTGAGCG TCGATTTTTG TGATGCTCGT CAGGGGGGCG GAGCCTATGGAAAAACGCCA GCAACGCGGC CTTTTTACGG TTCCTGGCCT TTTGCTGGCC TTTTGCTCACATGTTCTTTC CTGCGTTATC CCCTGATTCT GTGGATAACC GTATTACCGC CATGCAT