pEGFP-N3哺乳荧光质粒

基本信息

启动子:	CMV
复制子:	pUC ori
终止子:	SV40 poly(A) signal
质粒大小:	4729bp
原核抗性:	卡那霉素Kan
筛选标记:	遗传霉素G418
克隆菌株:	大肠杆菌DH5α
培养条件:	37℃，有氧，LB
表达宿主:	哺乳细胞
诱导方式:	无须诱导，瞬时表达
5'测序引物:	CMV-F：CGCAAATGGGCGGTAGGCGTG
3'测序引物:	根据序列设计引物

质粒简介

          pEGFP-N3 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-N3 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-N3 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 virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pEGFP-N3 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

质粒图谱

 

质粒序列

LOCUS       Exported                4729 bp ds-DNA    circular SYN 18-10-2015
FEATURES             Location/Qualifiers
     source          1..4729
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        61..364
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    609..665
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             675..1394
                     /codon_start=1
                     /product="enhanced GFP"
                     /note="EGFP"
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    1517..1638
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1645..2100)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2127..2231
                     /gene="bla"
                     /note="AmpR promoter"
     promoter        2233..2590
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      2441..2576
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             2625..3419
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="NeoR/KanR"
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3651..3698
                     /note="HSV TK poly(A) signal"
                     /note="herpesvirus thymidine kinase polyadenylation signal"
     rep_origin      4027..4615
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
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 gatccatcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      721 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      781 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      841 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
      901 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      961 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1021 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1081 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     1141 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     1201 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1261 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     1321 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1381 agctgtacaa gtaaagcggc cgcgactcta gatcataatc agccatacca catttgtaga
     1441 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     1501 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     1561 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     1621 actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     1681 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     1741 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     1801 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     1861 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     1921 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     1981 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     2041 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     2101 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2161 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2221 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     2281 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2341 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2401 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     2461 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     2521 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2581 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     2641 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     2701 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     2761 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     2821 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     2881 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     2941 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     3001 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     3061 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     3121 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     3181 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     3241 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     3301 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     3361 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     3421 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     3481 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     3541 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     3601 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     3661 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     3721 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     3781 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     3841 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     3901 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3961 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4021 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4081 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4141 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     4201 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4261 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4321 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4381 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4441 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4501 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4561 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4621 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4681 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcat