pEF1α-IRES-AcGFP1

pEF1α-IRES-AcGFP1

pEF1α-IRES-AcGFP1载体基本信息

pEF1α-IRES-AcGFP1载体质粒图谱和多克隆位点信息

pEF1α-IRES-AcGFP1载体简介

pEF1α-IRES-AcGFP1 is a bicistronic mammalian expression vector that allows the simultaneous, constitutive expression of a protein of interest and the green fluorescent protein AcGFP1. Expression of the bicistronic transcript is driven by the human elongation factor 1 alpha (EF1α) promoter, which continues to be constitutively active even after stable integration of the vector into the host cell genome. Stable expression of the bicistronic transcript allows the monitoring of a variety of cellular processes (such as differentiation in primary or stem cells), without the transgene silencing associated with CMV promoters.

In addition, the vector allows efficient flow cytometric detection of stably or transiently transfected mammalian cells expressing AcGFP1 and a protein of interest, without time-consuming drug and clonal selection.

pEF1α-IRES-AcGFP1 is designed to simultaneously and constitutively express a protein of interest and AcGFP1 in mammalian cells. AcGFP1 is a human-codon-optimized, monomeric green fluorescent protein derived from Aequorea coerulescens (the excitation and emission maxima of native AcGFP1 are 475 nm and 505 nm, respectively). Simultaneous expression of a protein of interest and AcGFP1 is made possible by the presence of an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES; 1, 2) positioned between the multiple cloning site (MCS) and the AcGFP1 gene. The IRES allows a protein of interest and AcGFP1 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 (3).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 (4). 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.

Location of Features

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

MCS (multiple cloning site): 1348–1422

IRES2 (internal ribosome entry site): 1423–2007

AcGFP1 (human-codon-optimized): 2011–2727

SV40 early polyA signal: 2883–2933

f1 origin of replication: 2980–3435 (complementary)

SV40 origin of replication: 3776–3911

PSV40e (SV40 early promoter and enhancer sequences): 3609–3877

Kanr/Neor(kanamycin/neomycin resistance gene): 3960–4754

HSV TK polyA signals: 4990–5008

pUC origin of replication: 5339–5982

Additional Information

pEF1α-IRES2-AcGFP1 can be used to quickly identify cells expressing a gene of interest by screening for AcGFP1 fluorescence. Genes inserted into the MCS must contain a start codon (ATG) and a stop codon. pEF1α-IRES2-AcGFP1 can be introduced into mammalian cells using any standard transfection method. Cells expressing AcGFP1can 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 of green-emitting cells. If required, stable transformants can be selected using G418 (4).

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 AcGFP1

Excitation: 475 nm

Emission: 505 nm

pEF1α-IRES-AcGFP1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 ATGGTGAGCA AGGGCGCCGA GCTGTTCACC

 2041 GGCATCGTGC CCATCCTGAT CGAGCTGAAT GGCGATGTGA ATGGCCACAA GTTCAGCGTG

 2101 AGCGGCGAGG GCGAGGGCGA TGCCACCTAC GGCAAGCTGA CCCTGAAGTT CATCTGCACC

 2161 ACCGGCAAGC TGCCTGTGCC CTGGCCCACC CTGGTGACCA CCCTGAGCTA CGGCGTGCAG

 2221 TGCTTCTCAC GCTACCCCGA TCACATGAAG CAGCACGACT TCTTCAAGAG CGCCATGCCT

 2281 GAGGGCTACA TCCAGGAGCG CACCATCTTC TTCGAGGATG ACGGCAACTA CAAGTCGCGC

 2341 GCCGAGGTGA AGTTCGAGGG CGATACCCTG GTGAATCGCA TCGAGCTGAC CGGCACCGAT

 2401 TTCAAGGAGG ATGGCAACAT CCTGGGCAAT AAGATGGAGT ACAACTACAA CGCCCACAAT

 2461 GTGTACATCA TGACCGACAA GGCCAAGAAT GGCATCAAGG TGAACTTCAA GATCCGCCAC

 2521 AACATCGAGG ATGGCAGCGT GCAGCTGGCC GACCACTACC AGCAGAATAC CCCCATCGGC

 2581 GATGGCCCTG TGCTGCTGCC CGATAACCAC TACCTGTCCA CCCAGAGCGC CCTGTCCAAG

 2641 GACCCCAACG AGAAGCGCGA TCACATGATC TACTTCGGCT TCGTGACCGC CGCCGCCATC

 2701 ACCCACGGCA TGGATGAGCT GTACAAGTGA GCGGCCGCGA CTCTAGATCA TAATCAGCCA

 2761 TACCACATTT GTAGAGGTTT TACTTGCTTT AAAAAACCTC CCACACCTCC CCCTGAACCT

 2821 GAAACATAAA ATGAATGCAA TTGTTGTTGT TAACTTGTTT ATTGCAGCTT ATAATGGTTA

 2881 CAAATAAAGC AATAGCATCA CAAATTTCAC AAATAAAGCA TTTTTTTCAC TGCATTCTAG

 2941 TTGTGGTTTG TCCAAACTCA TCAATGTATC TTAAGGCGTA AATTGTAAGC GTTAATATTT

 3001 TGTTAAAATT CGCGTTAAAT TTTTGTTAAA TCAGCTCATT TTTTAACCAA TAGGCCGAAA

 3061 TCGGCAAAAT CCCTTATAAA TCAAAAGAAT AGACCGAGAT AGGGTTGAGT GTTGTTCCAG

 3121 TTTGGAACAA GAGTCCACTA TTAAAGAACG TGGACTCCAA CGTCAAAGGG CGAAAAACCG

 3181 TCTATCAGGG CGATGGCCCA CTACGTGAAC CATCACCCTA ATCAAGTTTT TTGGGGTCGA

 3241 GGTGCCGTAA AGCACTAAAT CGGAACCCTA AAGGGAGCCC CCGATTTAGA GCTTGACGGG

 3301 GAAAGCCGGC GAACGTGGCG AGAAAGGAAG GGAAGAAAGC GAAAGGAGCG GGCGCTAGGG

 3361 CGCTGGCAAG TGTAGCGGTC ACGCTGCGCG TAACCACCAC ACCCGCCGCG CTTAATGCGC

 3421 CGCTACAGGG CGCGTCAGGT GGCACTTTTC GGGGAAATGT GCGCGGAACC CCTATTTGTT

 3481 TATTTTTCTA AATACATTCA AATATGTATC CGCTCATGAG ACAATAACCC TGATAAATGC

 3541 TTCAATAATA TTGAAAAAGG AAGAGTCCTG AGGCGGAAAG AACCAGCTGT GGAATGTGTG

 3601 TCAGTTAGGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA

 3661 TCTCAATTAG TCAGCAACCA GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT

 3721 GCAAAGCATG CATCTCAATT AGTCAGCAAC CATAGTCCCG CCCCTAACTC CGCCCATCCC

 3781 GCCCCTAACT CCGCCCAGTT CCGCCCATTC TCCGCCCCAT GGCTGACTAA TTTTTTTTAT

 3841 TTATGCAGAG GCCGAGGCCG CCTCGGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT

 3901 TTTTGGAGGC CTAGGCTTTT GCAAAGATCG ATCAAGAGAC AGGATGAGGA TCGTTTCGCA

 3961 TGATTGAACA AGATGGATTG CACGCAGGTT CTCCGGCCGC TTGGGTGGAG AGGCTATTCG

 4021 GCTATGACTG GGCACAACAG ACAATCGGCT GCTCTGATGC CGCCGTGTTC CGGCTGTCAG

 4081 CGCAGGGGCG CCCGGTTCTT TTTGTCAAGA CCGACCTGTC CGGTGCCCTG AATGAACTGC

 4141 AAGACGAGGC AGCGCGGCTA TCGTGGCTGG CCACGACGGG CGTTCCTTGC GCAGCTGTGC

 4201 TCGACGTTGT CACTGAAGCG GGAAGGGACT GGCTGCTATT GGGCGAAGTG CCGGGGCAGG

 4261 ATCTCCTGTC ATCTCACCTT GCTCCTGCCG AGAAAGTATC CATCATGGCT GATGCAATGC

 4321 GGCGGCTGCA TACGCTTGAT CCGGCTACCT GCCCATTCGA CCACCAAGCG AAACATCGCA

 4381 TCGAGCGAGC ACGTACTCGG ATGGAAGCCG GTCTTGTCGA TCAGGATGAT CTGGACGAAG

 4441 AGCATCAGGG GCTCGCGCCA GCCGAACTGT TCGCCAGGCT CAAGGCGAGC ATGCCCGACG

 4501 GCGAGGATCT CGTCGTGACC CATGGCGATG CCTGCTTGCC GAATATCATG GTGGAAAATG

 4561 GCCGCTTTTC TGGATTCATC GACTGTGGCC GGCTGGGTGT GGCGGACCGC TATCAGGACA

 4621 TAGCGTTGGC TACCCGTGAT ATTGCTGAAG AGCTTGGCGG CGAATGGGCT GACCGCTTCC

 4681 TCGTGCTTTA CGGTATCGCC GCTCCCGATT CGCAGCGCAT CGCCTTCTAT CGCCTTCTTG

 4741 ACGAGTTCTT CTGAGCGGGA CTCTGGGGTT CGAAATGACC GACCAAGCGA CGCCCAACCT

 4801 GCCATCACGA GATTTCGATT CCACCGCCGC CTTCTATGAA AGGTTGGGCT TCGGAATCGT

 4861 TTTCCGGGAC GCCGGCTGGA TGATCCTCCA GCGCGGGGAT CTCATGCTGG AGTTCTTCGC

 4921 CCACCCTAGG GGGAGGCTAA CTGAAACACG GAAGGAGACA ATACCGGAAG GAACCCGCGC

 4981 TATGACGGCA ATAAAAAGAC AGAATAAAAC GCACGGTGTT GGGTCGTTTG TTCATAAACG

 5041 CGGGGTTCGG TCCCAGGGCT GGCACTCTGT CGATACCCCA CCGAGACCCC ATTGGGGCCA

 5101 ATACGCCCGC GTTTCTTCCT TTTCCCCACC CCACCCCCCA AGTTCGGGTG AAGGCCCAGG

 5161 GCTCGCAGCC AACGTCGGGG CGGCAGGCCC TGCCATAGCC TCAGGTTACT CATATATACT

 5221 TTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA TCCTTTTTGA

 5281 TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC CACTGAGCGT CAGACCCCGT

 5341 AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG CGCGTAATCT GCTGCTTGCA

 5401 AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG GATCAAGAGC TACCAACTCT

 5461 TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA AATACTGTTC TTCTAGTGTA

 5521 GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT

 5581 AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG GGTTGGACTC

 5641 AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT CGTGCACACA

 5701 GCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC CTACAGCGTG AGCTATGAGA

 5761 AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT CCGGTAAGCG GCAGGGTCGG

 5821 AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT ATAGTCCTGT

 5881 CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG GGGGGCGGAG

 5941 CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC CTGGCCTTTT GCTGGCCTTT

 6001 TGCTCACATG TTCTTTCCTG CGTTATCCCC TGATTCTGTG GATAACCGTA TTACCGCCAT

 6061 GCAT

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