pEF1α-IRES-ZsGreen1

pEF1α-IRES-ZsGreen1

pEF1α-IRES-ZsGreen1载体基本信息

pEF1α-IRES-ZsGreen1载体质粒图谱和多克隆位点信息

pEF1α-IRES-ZsGreen1载体介绍

pEF1α-IRES-ZsGreen1 is designed to simultaneously and constitutively express a protein of interest and ZsGreen1 in mammalian cells. ZsGreen1 is a human-codon-optimized variant of the reef coral Zoanthus sp. green fluorescent protein (ZsGreen) that has been engineered for brighter fluorescence (1–3). The excitation and emission maxima of native ZsGreen1 are 493 nm and 505 nm, respectively. Simultaneous expression of a protein of interest and ZsGreen1 is made possible by the presence of an encephalomyocarditis virus internal ribosome entry site (IRES2; 4, 5) positioned between the multiple cloning site (MCS) and the ZsGreen1 gene. The IRES allows a protein of interest and ZsGreen1 to be translated from a single bicistronic mRNA. Stable, constitutive expression of the bicistronic transcript is driven by the EF1α promoter (PEF1α), which continues to be constitutively active even after vector integration into the host cell genome (6).

The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. This vector also has a neomycin-resistance cassette (Neor) that allows G418 selection of stably transfected eukaryotic cells (7).

This cassette consists of the SV40 early promoter (PSV40 e), a Tn5 kanamycin/neomycin resistance gene, and herpes simplex virus thymidine kinase (HSV TK) polyadenylation signals. A bacterial promoter upstream of the cassette drives expression of the kanamycin resistance gene in E. coli.

载体特征

PEF1α (human elongation factor 1 alpha promoter): 12–1346

MCS (multiple cloning site): 1348–1422

IRES2 (internal ribosome entry site): 1423–2007

ZsGreen1 (human-codon-optimized): 1999–2703

SV40 early polyA signal: 2859–2909

f1 origin of replication: 2956–3411 (complementary)

SV40 origin of replication: 3752–3887

PSV40e (SV40 early promoter and enhancer sequences): 3585–3853

Kanr/Neor (kanamycin/neomycin resistance gene): 3936–4730

HSV TK polyA signals: 4966–4984

pUC origin of replication: 5315–5958

补充信息

pEF1α-IRES2-ZsGreen1 can be used to quickly identify cells expressing a gene of interest by screening for ZsGreen1 fluorescence. Genes inserted into the MCS must contain a start codon (ATG) and a stop codon. pEF1α-IRES2-ZsGreen1 can be introduced into mammalian cells using any standard transfection method. Cells expressing ZsGreen1 can be detected by flow cytometry or fluorescence microscopy 24 hours after transfection. However, in some cases, up to 48 hours may be required for detection. If required, stable transformants can be selected using G418 (7).

Propagation in E. coli

Suitable host strains: DH5α 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) in E. coli hosts.

E. coli replication origin: pUC

Copy number: high

Excitation and Emission Maxima of ZsGreen1

Excitation: 493 nm

Emission: 505 nm

pEF1α-IRES-ZsGreen1载体序列

ORIGIN

    1 TAGTTATTAA TGAGTAATTC ATACAAAAGG ACTCGCCCCT GCCTTGGGGA ATCCCAGGGA

   61 CCGTCGTTAA ACTCCCACTA ACGTAGAACC CAGAGATCGC TGCGTTCCCG CCCCCTCACC

  121 CGCCCGCTCT CGTCATCACT GAGGTGGAGA AGAGCATGCG TGAGGCTCCG GTGCCCGTCA

  181 GTGGGCAGAG CGCACATCGC CCACAGTCCC CGAGAAGTTG GGGGGAGGGG TCGGCAATTG

  241 AACCGGTGCC TAGAGAAGGT GGCGCGGGGT AAACTGGGAA AGTGATGTCG TGTACTGGCT

  301 CCGCCTTTTT CCCGAGGGTG GGGGAGAACC GTATATAAGT GCAGTAGTCG CCGTGAACGT

  361 TCTTTTTCGC AACGGGTTTG CCGCCAGAAC ACAGGTAAGT GCCGTGTGTG GTTCCCGCGG

  421 GCCTGGCCTC TTTACGGGTT ATGGCCCTTG CGTGCCTTGA ATTACTTCCA CGCCCCTGGC

  481 TGCAGTACGT GATTCTTGAT CCCGAGCTTC GGGTTGGAAG TGGGTGGGAG AGTTCGAGGC

  541 CTTGCGCTTA AGGAGCCCCT TCGCCTCGTG CTTGAGTTGA GGCCTGGCTT GGGCGCTGGG

  601 GCCGCCGCGT GCGAATCTGG TGGCACCTTC GCGCCTGTCT CGCTGCTTTC GATAAGTCTC

  661 TAGCCATTTA AAATTTTTGA TGACCTGCTG CGACGCTTTT TTTCTGGCAA GATAGTCTTG

  721 TAAATGCGGG CCAAGATCTG CACACTGGTA TTTCGGTTTT TGGGGCCGCG GGCGGCGACG

  781 GGGCCCGTGC GTCCCAGCGC ACATGTTCGG CGAGGCGGGG CCTGCGAGCG CGGCCACCGA

  841 GAATCGGACG GGGGTAGTCT CAAGCTGGCC GGCCTGCTCT GGTGCCTGGC CTCGCGCCGC

  901 CGTGTATCGC CCCGCCCTGG GCGGCAAGGC TGGCCCGGTC GGCACCAGTT GCGTGAGCGG

  961 AAAGATGGCC GCTTCCCGGC CCTGCTGCAG GGAGCTCAAA ATGGAGGACG CGGCGCTCGG

 1021 GAGAGCGGGC GGGTGAGTCA CCCACACAAA GGAAAAGGGC CTTTCCGTCC TCAGCCGTCG

 1081 CTTCATGTGA CTCCACGGAG TACCGGGCGC CGTCCAGGCA CCTCGATTAG TTCTCGAGCT

 1141 TTTGGAGTAC GTCGTCTTTA GGTTGGGGGG AGGGGTTTTA TGCGATGGAG TTTCCCCACA

 1201 CTGAGTGGGT GGAGACTGAA GTTAGGCCAG CTTGGCACTT GATGTAATTC TCCTTGGAAT

 1261 TTGCCCTTTT TGAGTTTGGA TCTTGGTTCA TTCTCAAGCC TCAGACAGTG GTTCAAAGTT

 1321 TTTTTCTTCC ATTTCAGGTG TCGTGACGCT AGCGCTACCG GACTCAGATC TCGAGCTCAA

 1381 GCTTCGAATT CTGCAGTCGA CGGTACCGCG GGCCCGGGAT CCGCCCCTCT CCCTCCCCCC

 1441 CCCCTAACGT TACTGGCCGA AGCCGCTTGG AATAAGGCCG GTGTGCGTTT GTCTATATGT

 1501 TATTTTCCAC CATATTGCCG TCTTTTGGCA ATGTGAGGGC CCGGAAACCT GGCCCTGTCT

 1561 TCTTGACGAG CATTCCTAGG GGTCTTTCCC CTCTCGCCAA AGGAATGCAA GGTCTGTTGA

 1621 ATGTCGTGAA GGAAGCAGTT CCTCTGGAAG CTTCTTGAAG ACAAACAACG TCTGTAGCGA

 1681 CCCTTTGCAG GCAGCGGAAC CCCCCACCTG GCGACAGGTG CCTCTGCGGC CAAAAGCCAC

 1741 GTGTATAAGA TACACCTGCA AAGGCGGCAC AACCCCAGTG CCACGTTGTG AGTTGGATAG

 1801 TTGTGGAAAG AGTCAAATGG CTCTCCTCAA GCGTATTCAA CAAGGGGCTG AAGGATGCCC

 1861 AGAAGGTACC CCATTGTATG GGATCTGATC TGGGGCCTCG GTACACATGC TTTACATGTG

 1921 TTTAGTCGAG GTTAAAAAAA CGTCTAGGCC CCCCGAACCA CGGGGACGTG GTTTTCCTTT

 1981 GAAAAACACG ATGATAATAT GGCCACAACC ATGGCCCAGT CCAAGCACGG CCTGACCAAG

 2041 GAGATGACCA TGAAGTACCG CATGGAGGGC TGCGTGGACG GCCACAAGTT CGTGATCACC

 2101 GGCGAGGGCA TCGGCTACCC CTTCAAGGGC AAGCAGGCCA TCAACCTGTG CGTGGTGGAG

 2161 GGCGGCCCCT TGCCCTTCGC CGAGGACATC TTGTCCGCCG CCTTCATGTA CGGCAACCGC

 2221 GTGTTCACCG AGTACCCCCA GGACATCGTC GACTACTTCA AGAACTCCTG CCCCGCCGGC

 2281 TACACCTGGG ACCGCTCCTT CCTGTTCGAG GACGGCGCCG TGTGCATCTG CAACGCCGAC

 2341 ATCACCGTGA GCGTGGAGGA GAACTGCATG TACCACGAGT CCAAGTTCTA CGGCGTGAAC

 2401 TTCCCCGCCG ACGGCCCCGT GATGAAGAAG ATGACCGACA ACTGGGAGCC CTCCTGCGAG

 2461 AAGATCATCC CCGTGCCCAA GCAGGGCATC TTGAAGGGCG ACGTGAGCAT GTACCTGCTG

 2521 CTGAAGGACG GTGGCCGCTT GCGCTGCCAG TTCGACACCG TGTACAAGGC CAAGTCCGTG

 2581 CCCCGCAAGA TGCCCGACTG GCACTTCATC CAGCACAAGC TGACCCGCGA GGACCGCAGC

 2641 GACGCCAAGA ACCAGAAGTG GCACCTGACC GAGCACGCCA TCGCCTCCGG CTCCGCCTTG

 2701 CCCTGAGCGG CCGCGACTCT AGATCATAAT CAGCCATACC ACATTTGTAG AGGTTTTACT

 2761 TGCTTTAAAA AACCTCCCAC ACCTCCCCCT GAACCTGAAA CATAAAATGA ATGCAATTGT

 2821 TGTTGTTAAC TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA GCATCACAAA

 2881 TTTCACAAAT AAAGCATTTT TTTCACTGCA TTCTAGTTGT GGTTTGTCCA AACTCATCAA

 2941 TGTATCTTAA GGCGTAAATT GTAAGCGTTA ATATTTTGTT AAAATTCGCG TTAAATTTTT

 3001 GTTAAATCAG CTCATTTTTT AACCAATAGG CCGAAATCGG CAAAATCCCT TATAAATCAA

 3061 AAGAATAGAC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT CCACTATTAA

 3121 AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT GGCCCACTAC

 3181 GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA CTAAATCGGA

 3241 ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC GTGGCGAGAA

 3301 AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA GCGGTCACGC

 3361 TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG TCAGGTGGCA

 3421 CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA CATTCAAATA

 3481 TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA AAAAGGAAGA

 3541 GTCCTGAGGC GGAAAGAACC AGCTGTGGAA TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC

 3601 AGGCTCCCCA GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCAGGTG

 3661 TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC

 3721 AGCAACCATA GTCCCGCCCC TAACTCCGCC CATCCCGCCC CTAACTCCGC CCAGTTCCGC

 3781 CCATTCTCCG CCCCATGGCT GACTAATTTT TTTTATTTAT GCAGAGGCCG AGGCCGCCTC

 3841 GGCCTCTGAG CTATTCCAGA AGTAGTGAGG AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA

 3901 AGATCGATCA AGAGACAGGA TGAGGATCGT TTCGCATGAT TGAACAAGAT GGATTGCACG

 3961 CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA

 4021 TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG

 4081 TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAAGA CGAGGCAGCG CGGCTATCGT

 4141 GGCTGGCCAC GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CGTTGTCACT GAAGCGGGAA

 4201 GGGACTGGCT GCTATTGGGC GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC

 4261 CTGCCGAGAA AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG

 4321 CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT ACTCGGATGG

 4381 AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG

 4441 AACTGTTCGC CAGGCTCAAG GCGAGCATGC CCGACGGCGA GGATCTCGTC GTGACCCATG

 4501 GCGATGCCTG CTTGCCGAAT ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT

 4561 GTGGCCGGCT GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG

 4621 CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT ATCGCCGCTC

 4681 CCGATTCGCA GCGCATCGCC TTCTATCGCC TTCTTGACGA GTTCTTCTGA GCGGGACTCT

 4741 GGGGTTCGAA ATGACCGACC AAGCGACGCC CAACCTGCCA TCACGAGATT TCGATTCCAC

 4801 CGCCGCCTTC TATGAAAGGT TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT

 4861 CCTCCAGCGC GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCTAGGGGGA GGCTAACTGA

 4921 AACACGGAAG GAGACAATAC CGGAAGGAAC CCGCGCTATG ACGGCAATAA AAAGACAGAA

 4981 TAAAACGCAC GGTGTTGGGT CGTTTGTTCA TAAACGCGGG GTTCGGTCCC AGGGCTGGCA

 5041 CTCTGTCGAT ACCCCACCGA GACCCCATTG GGGCCAATAC GCCCGCGTTT CTTCCTTTTC

 5101 CCCACCCCAC CCCCCAAGTT CGGGTGAAGG CCCAGGGCTC GCAGCCAACG TCGGGGCGGC

 5161 AGGCCCTGCC ATAGCCTCAG GTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT

 5221 TTAATTTAAA AGGATCTAGG TGAAGATCCT TTTTGATAAT CTCATGACCA AAATCCCTTA

 5281 ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAA AAGATCAAAG GATCTTCTTG

 5341 AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTGCAAACA AAAAAACCAC CGCTACCAGC

 5401 GGTGGTTTGT TTGCCGGATC AAGAGCTACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG

 5461 CAGAGCGCAG ATACCAAATA CTGTTCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA

 5521 GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC

 5581 CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGA CGATAGTTAC CGGATAAGGC

 5641 GCAGCGGTCG GGCTGAACGG GGGGTTCGTG CACACAGCCC AGCTTGGAGC GAACGACCTA

 5701 CACCGAACTG AGATACCTAC AGCGTGAGCT ATGAGAAAGC GCCACGCTTC CCGAAGGGAG

 5761 AAAGGCGGAC AGGTATCCGG TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT

 5821 TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA

 5881 GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA TGGAAAAACG CCAGCAACGC

 5941 GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT CACATGTTCT TTCCTGCGTT

 6001 ATCCCCTGAT TCTGTGGATA ACCGTATTAC CGCCATGCAT

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