pCMS-EGFP

pCMS-EGFP is a mammalian expression vector that allows you to express your gene of interest at high levels by cloning it into a multiple cloning site (MCS) downstream of the immediate early promoter of cytomegalovirus (PCMV IE). The intervening sequence (IVS) between PCMV IE and the MCS is an intron that is efficiently spliced out following transcription. SV40 polyadenylation signals downstream of the MCS direct proper processing of the 3' end of the mRNA from your gene of interest. Bacteriophage T7 and T3 promoters are located upstream and downstream of the MCS, respectively.

pCMS-EGFP uses the enhanced green flourescent protein (EGFP) as a transformation marker. EGFP is a red-shifted variant of wild-type GFP from Aquorea victoria (1–3). EGFP has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.) EGFP 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. EGFP is expressed from the SV40 enhancer/promoter, and a polyadenylation signal from the bovine growth hormone (BGH) gene directs proper processing of the 3' end of the EGFP mRNA. The SV40 origin also allows for replication in mammalian cells expressing the SV40 T antigen.The vector backbone also contains the β-lactamase gene for ampicillin resistance and a ColE1 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

The name of the vector comes from the order of four critical elements: the CMV IE promoter; the MCS; the SV40 promoter; and EGFP.

载体应用

Genes cloned into the MCS must contain an ATG start codon. pCMS-EGFP and derivatives can be introduced into mammalian cells by any standard method. Transfected cells can be monitored and/or selected by flow cytometry based on the fluorescence of EGFP. Sense or antisense RNA can be transcribed from the T7 and T3 promoters, respectively.

0001 TCAATATTGG CCATTAGCCA TATTATTCAT TGGTTATATA GCATAAATCA ATATTGGCTA 
0061 TTGGCCATTG CATACGTTGT ATCTATATCA TAATATGTAC ATTTATATTG GCTCATGTCC 
0121 AATATGACCG CCATGTTGGC ATTGATTATT GACTAGTTAT TAATAGTAAT CAATTACGGG 
0181 GTCATTAGTT CATAGCCCAT ATATGGAGTT CCGCGTTACA TAACTTACGG TAAATGGCCC 
0241 GCCTGGCTGA CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT 
0301 AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC 
0361 CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTCCG CCCCCTATTG ACGTCAATGA 
0421 CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC TTACGGGACT TTCCTACTTG 
0481 GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAC 
0541 CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT 
0601 CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTG 
0661 CGATCGCCCG CCCCGTTGAC GCAAATGGGC GGTAGGCGTG TACGGTGGGA GGTCTATATA 
0721 AGCAGAGCTC GTTTAGTGAA CCGTCAGATC ACTAGAAGCT TTATTGCGGT AGTTTATCAC 
0781 AGTTAAATTG CTAACGCAGT CAGTGCTTCT GACACAACAG TCTCGAACTT AAGCTGCAGT 
0841 GACTCTCTTA AGGTAGCCTT GCAGAAGTTG GTCGTGAGGC ACTGGGCAGG TAAGTATCAA 
0901 GGTTACAAGA CAGGTTTAAG GAGACCAATA GAAACTGGGC TTGTCGAGAC AGAGAAGACT 
0961 CTTGCGTTTC TGATAGGCAC CTATTGGTCT TACTGACATC CACTTTGCCT TTCTCTCCAC 
1021 AGGTGTCCAC TCCCAGTTCA ATTACAGCTC TTAAGGCTAG AGTACTTAAT ACGACTCACT 
1081 ATAGGCTAGC CTCGAGAATT CACGCGTGGT ACCTCTAGAG TCGACCCGGG CGGCCGCTTC 
1141 CCTTTAGTGA GGGTTAATGC TTCGCAGACA TGATAAGATA CATTGATGAG TTTGGACAAA 
1201 CCACAACTAG AATGCAGTGA AAAAAATGCT TTATTTGTGA AATTTGTGAT GCTATTGCTT 
1261 TATTTGTAAC CATTATAAGC TGCAATAAAC AAGTTAACAA CAACAATTGC ATTCATTTTA 
1321 TGTTTCAGGT TCAGGGGGAG ATGTGGGAGG TTTTTTAAAG CAAGTAAAAC CTCTACAAAT 
1381 GTGGTAAAAT CCGATAAGGA TCGATCCGGG CTGGCGTAAT AGCGAAGAGG CCCGCACCGA 
1441 TCGCCCTTCC CAACAGTTGC GCAGCCTGAA TGGCGAATGG ACGCGCCCTG TAGCGGCGCA 
1501 TTAAGCGCGG CGGGTGTGGT GGTTACGCGC AGCGTGACCG CTACACTTGC CAGCGCCCTA 
1561 GCGCCCGCTC CTTTCGCTTT CTTCCCTTCC TTTCTCGCCA CGTTCGCCGG CTTTCCCCGT 
1621 CAAGCTCTAA ATCGGGGGCT CCCTTTAGGG TTCCGATTTA GAGCTTTACG GCACCTCGAC 
1681 CGCAAAAAAC TTGATTTGGG TGATGGTTCA CGTAGTGGGC CATCGCCCTG ATAGACGGTT 
1741 TTTCGCCCTT TGACGTTGGA GTCCACGTTC TTTAATAGTG GACTCTTGTT CCAAACTGGA 
1801 ACAACACTCA ACCCTATCTC GGTCTATTCT TTTGATTTAT AAGGGATTTT GCCGATTTCG 
1861 GCCTATTGGT TAAAAAATGA GCTGATTTAA CAAATATTTA ACGCGAATTT TAACAAAATA 
1921 TTAACGTTTA CAATTTCGCC TGATGCGGTA TTTTCTCCTT ACGCATCTGT GCGGTATTTC 
1981 ACACCGCATA CGCGGATCTG CGCAGCACCA TGGCCTGAAA TAACCTCTGA AAGAGGAACT 
2041 TGGTTAGGTA CCTTCTGAGG CGGAAAGAAC CAGCTGTGGA ATGTGTGTCA GTTAGGGTGT 
2101 GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA 
2161 GCAACCAGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCAT 
2221 CTCAATTAGT CAGCAACCAT AGTCCCGCCC CTAACTCCGC CCATCCCGCC CCTAACTCCG 
2281 CCCAGTTCCG CCCATTCTCC GCCCCATGGC TGACTAATTT TTTTTATTTA TGCAGAGGCC 
2341 GAGGCCGCCT CGGCCTCTGA GCTATTCCAG AAGTAGTGAG GAGGCTTTTT TGGAGGCCTA 
2401 GGCTTTTGCA AAAAGCTTGC CACAACCCGG GATCCACCGG TCGCCACCAT GGTGAGCAAG 
2461 GGCGAGGAGC TGTTCACCGG GGTGGTGCCC ATCCTGGTCG AGCTGGACGG CGACGTAAAC 
2521 GGCCACAAGT TCAGCGTGTC CGGCGAGGGC GAGGGCGATG CCACCTACGG CAAGCTGACC 
2581 CTGAAGTTCA TCTGCACCAC CGGCAAGCTG CCCGTGCCCT GGCCCACCCT CGTGACCACC 
2641 CTGACCTACG GCGTGCAGTG CTTCAGCCGC TACCCCGACC ACATGAAGCA GCACGACTTC 
2701 TTCAAGTCCG CCATGCCCGA AGGCTACGTC CAGGAGCGCA CCATCTTCTT CAAGGACGAC 
2761 GGCAACTACA AGACCCGCGC CGAGGTGAAG TTCGAGGGCG ACACCCTGGT GAACCGCATC 
2821 GAGCTGAAGG GCATCGACTT CAAGGAGGAC GGCAACATCC TGGGGCACAA GCTGGAGTAC 
2881 AACTACAACA GCCACAACGT CTATATCATG GCCGACAAGC AGAAGAACGG CATCAAGGTG 
2941 AACTTCAAGA TCCGCCACAA CATCGAGGAC GGCAGCGTGC AGCTCGCCGA CCACTACCAG 
3001 CAGAACACCC CCATCGGCGA CGGCCCCGTG CTGCTGCCCG ACAACCACTA CCTGAGCACC 
3061 CAGTCCGCCC TGAGCAAAGA CCCCAACGAG AAGCGCGATC ACATGGTCCT GCTGGAGTTC 
3121 GTGACCGCCG CCGGGATCAC TCTCGGCATG GACGAGCTGT ACAAGTAAAG CGGCCCTAGA 
3181 GCTCGCTGAT CAGCCTCGAC TGTGCCTTCT AGTTGCCAGC CATCTGTTGT TTGCCCCTCC 
3241 CCCGTGCCTT CCTTGACCCT GGAAGGTGCC ACTCCCACTG TCCTTTCCTA ATAAAATGAG 
3301 GAAATTGCAT CGCATTGTCT GAGTAGGTGT CATTCTATTC TGGGGGGTGG GGTGGGGCAG 
3361 GACAGCAAGG GGGAGGATTG GGAAGACAAT AGCAGGCATG CTGGGGATGC GGTGGGCTCT 
3421 ATGGCTTCTG AGGCGGAAAG AACCAGCTGG GGCAGATCCG CGTATGGTGC ACTCTCAGTA 
3481 CAATCTGCTC TGATGCCGCA TAGTTAAGCC AGCCCCGACA CCCGCCAACA CCCGCTGACG 
3541 CGCCCTGACG GGCTTGTCTG CTCCCGGCAT CCGCTTACAG ACAAGCTGTG ACCGTCTCCG 
3601 GGAGCTGCAT GTGTCAGAGG TTTTCACCGT CATCACCGAA ACGCGCGAGA CGAAAGGGCC 
3661 TCGTGATACG CCTATTTTTA TAGGTTAATG TCATGATAAT AATGGTTTCT TAGACGTCAG 
3721 GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC TAAATACATT 
3781 CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA 
3841 GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTTTTTT GCGGCATTTT 
3901 GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT GAAGATCAGT 
3961 TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC CTTGAGAGTT 
4021 TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA AGTTCTGCTA TGTGGCGCGG 
4081 TATTATCCCG TATTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA 
4141 ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA 
4201 GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC TTACTTCTGA 
4261 CAACGATCGG AGGACCGAAG GAGCTAACCG CTTTTTTGCA CAACATGGGG GATCATGTAA 
4321 CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC GAGCGTGACA 
4381 CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA 
4441 CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT GCAGGACCAC 
4501 TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA GCCGGTGAGC 
4561 GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC CGTATCGTAG 
4621 TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG ATCGCTGAGA 
4681 TAGGTGCCTC ACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT 
4741 AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA 
4801 ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG 
4861 AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA 
4921 CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT 
4981 TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC 
5041 CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA 
5101 TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA 
5161 GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC 
5221 CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA 
5281 GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA 
5341 CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG 
5401 GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC 
5461 TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG 
5521 CTCACATGGC TCGACAGATC T