pCDF1-MCS2-EF1-Puro

pCDF1-MCS2-EF1-Puro

pCDF1-MCS2-EF1-Puro载体基本信息：

pCDF1-MCS2-EF1-Puro载体质粒图谱和多克隆位点信息：

pCDF1-MCS2-EF1-Puro载体简介：

背景简介：

A. Purpose of this Manual

This manual provides details and information necessary to generate expression constructs of your gene of interest in the pCDF lentivectors. Specifically, it provides critical instructions on amplification and cloning the cDNA into the pCDF Vectors, and verifying final expression constructs. This manual does not include information on packaging the pCDF expression constructs into pseudotyped viral particles or transducing your target cells of choice with these particles.

B. Advantages of the Lentivector Expression System Lentiviral expression vectors are the most effective vehicles for delivering and expression of a gene of interest to almost any mammalian cell—including non-dividing cells and model organisms (C.A. Machida, 2003; M. Federico, 2003; W. C. Heiser, 2004). As with standard plasmid vectors, it is possible to introduce lentivector expression constructs in plasmid form into the cells with low-tomedium efficiency using conventional transfection protocols.

However, by packaging the lentivector construct into viral particles, you can obtain highly efficient transduction of expression constructs—even with the most difficult to transfect cells, such as primary, stem, and differentiated cells. The expression construct transduced in target cells is integrated into genomic DNA and provides stable, long-term expression of the target gene.

The lentiviral cDNA expression system consists of three main components:

(1) The lentiviral expression vector (e.g., pCDF1-MCS2-EF1-Puro)

(2) The lentiviral packaging plasmids (e.g., pPACKF1 Packaging Plasmid mix)

(3) A pseudoviral particle producer cell line (e.g., 293TN cells)

The expression lentivector contains the genetic elements responsible for packaging, transduction, stable integration of the viral expression construct into genomic DNA, and expression of the target gene sequence. The packaging vector provides all the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant viral particles. To produce a high titer of viral particles, expression and packaging vectors are transiently cotransfected into producer mammalian cells (e.g., HEK 293 cells). For a detailed description of SBI’s Lentivector expression system, please refer to the Lentivector Expression Systems user manual.

SBI’s novel pCDF Vectors are derived from feline immunodeficiency virus (FIV; Poeschla, 2003; for Safety Guidelines when working with these vectors, see section G). These pCDF Vectors, developed at SBI, are self-inactivating as a result of a deletion in the U3 region of 3’ ΔLTR (see Appendix for Vector Features). Upon integration into the genome, the 5’ LTR promoter is inactivated, which prevents formation of replication-competent viral particles.

When expressed, the hybrid CMV/FIV 5’ LTR drives high level transcription of the viral construct and produces a transcript that contains all the necessary functional elements (i.e., Psi, RRE, and cPPT) for efficient packaging. When this construct is expressed in HEK 293 cells that also express viral coat proteins (i.e., a packaging cell line), the pCDF transcripts are efficiently packaged into pseudoviral particles. After isolation, these pseudoviral particles containing the RNA version of the pCDF expression cassette can be efficiently transduced into any mammalian target cells. Following transduction into the target cells, this expression cassette is reverse transcribed and integrated into the genome of the target cell. The pCDF Vectors also contain a bacterial origin of replication and ampicillin resistance (AmpR) gene for propagation and selection in E.coli.

The pCDF1-MCS2-EF1-Puro Vector (Cat. # CD110B-1) contains a puromycin resistance gene, under the control of a constitutive EF1 promoter and a WPRE regulatory element, to enable selection of target cells stably expressing the cDNA template. The pCDF1-MCS2-EF1-copGFP Vector (Cat. # CD111B-1) contains a copGFP gene under the control of a EF1 promoter and WPRE element. CopGFP is a novel fluorescent protein ,derived from copepod plankton (Panalina sp.), which is similar to EGFP but has a brighter color This gene serves as a reporter for the transfected or transduced cells.

pCDF Cloning and Expression Lentivectors

The FIV derived pCDF vectors contain the following features:

 CMV promoter—promotes a high level of expression of your gene of interest in a wide variety of cell lines.

 Multiple Cloning Site (MCS)—for cloning the gene of interest in MCS located downstream of CMV promoter.

 WPRE element—enhances stability and translation of the CMVdriven transcripts.

 SV40 polyadenylation signal—enables efficient termination of transcription and processing of recombinant transcripts.

Optional second expression cassette—provides expression of puromycin resistance gene or copGFP reporter under control of constitutive elongation factor 1 (EF1) promoter for selection or FACS analysis of transduced cells.

 Hybrid CMV-5LTR promoter—provides a high level of expression of the full-length viral transcript in producer 293 cells.

 Genetic elements (cPPT, GAG, LTRs)—necessary for packaging, transducing, and stably integrating the viral expression construct into genomic DNA.

 SV40 origin—for stable propagation of the pCDF plasmid in mammalian cells.

 pUC origin—for high copy replication and maintenance of the plasmid in E.coli cells.

 Ampicillin resistance gene—for selection in E.coli cells.

pCDF1-MCS2-EF1-Puro载体序列：

ORIGIN

    1 CATATGCCAA GTACGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC CTGGCATTAT

   61 GCCCAGTACA TGACCTTATG GGACTTTCCT ACTTGGCAGT ACATCTACGT ATTAGTCATC

  121 GCTATTACCA TGGTGATGCG GTTTTGGCAG TACATCAATG GGCGTGGATA GCGGTTTGAC

  181 TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA

  241 AATCAACGGG ACTTTCCAAA ATGTCGTAAC AACTCCGCCC CATTGACGCA AATGGGCGGT

  301 AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTTGTG AAACTTCGAG GAGTCTCTTT

  361 GTTGAGGACT TTTGAGTTCT CCCTTGAGGC TCCCACAGAT ACAATAAATA TTTGAGATTG

  421 AACCCTGTCG AGTATCTGTG TAATCTTTTT TACCTGTGAG GTCTCGGAAT CCGGGCCGAG

  481 AACTTCGCAG TTGGCGCCCG AACAGGGACT TGATTGAGAG TGATTGAGGA AGTGAAGCTA

  541 GAGCAATAGA AAGCTGTTAA GCAGAACTCC TGCTGACCTA AATAGGGAAG CAGTAGCAGA

  601 CGCTGCTAAC AGTGAGTATC TCTAGTGAAG CAGACTCGAG CTCATAATCA AGTCATTGTT

  661 TAAAGGCCCA GATAAATTAC ATCTGGTGAC TCTTCGCGGA CCTTCAAGCC AGGAGATTCG

  721 CCGAGGGACA GTCAACAAGG TAGGAGAGAT TCTACAGCAA CATGGGGAAT GGACAGGGGC

  781 GAGATTGGAA AATGGCCATT AAGAGATGTA GTAATGTTGC TGTAGGAGTA GGGGGGAAGA

  841 GTAAAAAATT TGGAGAAGGG AATTTCAGAT GGGCCATTAG AATGGCTAAT GTATCTACAG

  901 GACGAGAACC TGGTGATATA CCAGAGACTT TAGATCAACT AAGGTTGGTT ATTTGCGATT

  961 TACAAGAAAG AAGAGAAAAA TTTGGATCTA GCAAAGAAAT TGATATGGCA ATTCCTGCAT

 1021 TGAGGAGAAA TGGTAGGCAA TGTGGCATGT CTGAAAAAGA GGAGGAATGA TGAAGTATCT

 1081 CAGACTTATT TTATAAGGGA GATACTGTGC TGAGTTCTTC CCTTTGAGGA AGGTATGTCA

 1141 TATCCTAGAC ATAGTCTCAA TTTTAAAAGA AGAGGTAGGA TAGGAGGGAT GGCCCCTTAT

 1201 GAATTATTAG CACAACAAGA ATCCTTAAGA ATACAAGATT ATTTTTCTGC AATACCACAA

 1261 AAATTGCAAG CACAGTGGAT TTATTATAAA GATCAAAAAG ATAAGAAATG GAAAGGACCA

 1321 ATGAGAGTAG AATACTGGGG ACAGGGATCA GTATTATTAA AGGATGAAGA GAAGGGATAT

 1381 TTTCTTATAA TCGATACTAG TATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT

 1441 GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT GATGCGGTTT TGGCAGTACA

 1501 TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC AAGTCTCCAC CCCATTGACG

 1561 TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT CGTAACAACT

 1621 CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG

 1681 CTCGTTTAGT GAACCGTCAG ATCGCCTGGA GACGCCATCC ACGCTGTTTT GACCTCCATA

 1741 GAAGATTCTA GAGCCCGGGC GCGCCGGATC CAGATCTTAA TTAATTTAAA TGAATTCGCG

 1801 GCCGCGAAGG ATCTGCGATC GCTCCGGTGC CCGTCAGTGG GCAGAGCGCA CATCGCCCAC

 1861 AGTCCCCGAG AAGTTGGGGG GAGGGGTCGG CAATTGAACG GGTGCCTAGA GAAGGTGGCG

 1921 CGGGGTAAAC TGGGAAAGTG ATGTCGTGTA CTGGCTCCGC CTTTTTCCCG AGGGTGGGGG

 1981 AGAACCGTAT ATAAGTGCAG TAGTCGCCGT GAACGTTCTT TTTCGCAACG GGTTTGCCGC

 2041 CAGAACACAG CTGAAGCTTC GAGGGGCTCG CATCTCTCCT TCACGCGCCC GCCGCCCTAC

 2101 CTGAGGCCGC CATCCACGCC GGTTGAGTCG CGTTCTGCCG CCTCCCGCCT GTGGTGCCTC

 2161 CTGAACTGCG TCCGCCGTCT AGGTAAGTTT AAAGCTCAGG TCGAGACCGG GCCTTTGTCC

 2221 GGCGCTCCCT TGGAGCCTAC CTAGACTCAG CCGGCTCTCC ACGCTTTGCC TGACCCTGCT

 2281 TGCTCAACTC TACGTCTTTG TTTCGTTTTC TGTTCTGCGC CGTTACAGAT CCAAGCTGTG

 2341 ACCGGCGCCT ACGCTAGATG ACCGAGTACA AGCCCACGGT GCGCCTCGCC ACCCGCGACG

 2401 ACGTCCCCAG GGCCGTACGC ACCCTCGCCG CCGCGTTCGC CGACTACCCC GCCACGCGCC

 2461 ACACCGTCGA TCCGGACCGC CACATCGAGC GGGTCACCGA GCTGCAAGAA CTCTTCCTCA

 2521 CGCGCGTCGG GCTCGACATC GGCAAGGTGT GGGTCGCGGA CGACGGCGCC GCGGTGGCGG

 2581 TCTGGACCAC GCCGGAGAGC GTCGAAGCGG GGGCGGTGTT CGCCGAGATC GGCCCGCGCA

 2641 TGGCCGAGTT GAGCGGTTCC CGGCTGGCCG CGCAGCAACA GATGGAAGGC CTCCTGGCGC

 2701 CGCACCGGCC CAAGGAGCCC GCGTGGTTCC TGGCCACCGT CGGCGTCTCG CCCGACCACC

 2761 AGGGCAAGGG TCTGGGCAGC GCCGTCGTGC TCCCCGGAGT GGAGGCGGCC GAGCGCGCCG

 2821 GGGTGCCCGC CTTCCTGGAG ACCTCCGCGC CCCGCAACCT CCCCTTCTAC GAGCGGCTCG

 2881 GCTTCACCGT CACCGCCGAC GTCGAGGTGC CCGAAGGACC GCGCACCTGG TGCATGACCC

 2941 GCAAGCCCGG TGCCTGAGTC GACAATCAAC CTCTGGATTA CAAAATTTGT GAAAGATTGA

 3001 CTGGTATTCT TAACTATGTT GCTCCTTTTA CGCTATGTGG ATACGCTGCT TTAATGCCTT

 3061 TGTATCATGC TATTGCTTCC CGTATGGCTT TCATTTTCTC CTCCTTGTAT AAATCCTGGT

 3121 TGCTGTCTCT TTATGAGGAG TTGTGGCCCG TTGTCAGGCA ACGTGGCGTG GTGTGCACTG

 3181 TGTTTGCTGA CGCAACCCCC ACTGGTTGGG GCATTGCCAC CACCTGTCAG CTCCTTTCCG

 3241 GGACTTTCGC TTTCCCCCTC CCTATTGCCA CGGCGGAACT CATCGCCGCC TGCCTTGCCC

 3301 GCTGCTGGAC AGGGGCTCGG CTGTTGGGCA CTGACAATTC CGTGGTGTTG TCGGGGAAAT

 3361 CATCGTCCTT TCCTTGGCTG CTCGCCTGTG TTGCCACCTG GATTCTGCGC GGGACGTCCT

 3421 TCTGCTACGT CCCTTCGGCC CTCAATCCAG CGGACCTTCC TTCCCGCGGC CTGCTGCCGG

 3481 CTCTGCGGCC TCTTCCGCGT CTTCGCCTTC GCCCTCAGAC GAGTCGGATC TCCCTTTGGG

 3541 CCGCCTCCCC GCCTGGGTAC CGATGACAGA GTTAGAAGAT CGCTTCAGGA AGCTATTTGG

 3601 CACGACTTCT ACAACGGGAG ACAGCACAGT AGATTCTGAA GATGAACCTC CTAAAAAAGA

 3661 AAAAAGGGTG GACTGGGATG AGTATTGGAA CCCTGAAATC GATAGCTTCC AGTGCTTTGT

 3721 GAAACTTCGA GGAGTCTCTT TGTTGAGGAC TTTTGAGTTC TCCCTTGAGG CTCCCACAGA

 3781 TACAATAAAT ATTTGAGATT GAACCCTGTC GAGTATCTGT GTAATCTTTT TTACCTGTGA

 3841 GGTCTCGGAA TCCGGGCCGA GAACTTCGCA GCGAGCTCAT TGTACCGCGA ACTTGTTTAT

 3901 TGCAGCTTAT AATGGTTACA AATAAAGCAA TAGCATCACA AATTTCACAA ATAAAGCATT

 3961 TTTTTCACTG CATTCTAGTT GTGGTTTGTC CAAACTCATC AATGTATCTT ATCATGTCTG

 4021 GCTCTAGCTA TCCCGCCCCT AACTCCGCCC AGTTCCGCCC ATTCTCCGCC CCATGGCTGA

 4081 CTAATTTTTT TTATTTATGC AGAGGCCGAG GCCGCCTCGG CCTCTGAGCT ATTCCAGAAG

 4141 TAGTGAGGAG GCTTTTTTGG AGGCCTAGAC TTTTGCAGAG ACGGCCCAAA TTCGTAATCA

 4201 TGGTCATAGC TGTTTCCTGT GTGAAATTGT TATCCGCTCA CAATTCCACA CAACATACGA

 4261 GCCGGAAGCA TAAAGTGTAA AGCCTGGGGT GCCTAATGAG TGAGCTAACT CACATTAATT

 4321 GCGTTGCGCT CACTGCCCGC TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAATGA

 4381 ATCGGCCAAC GCGCGGGGAG AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC

 4441 ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG

 4501 GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC

 4561 CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC

 4621 CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA

 4681 CTATAAAGAT ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC

 4741 CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT

 4801 AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG

 4861 CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC

 4921 AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA

 4981 GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT

 5041 AGAAGGACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT

 5101 GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG

 5161 CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG

 5221 TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA

 5281 AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA

 5341 TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG

 5401 ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA

 5461 CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG

 5521 GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT

 5581 GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT

 5641 TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC

 5701 TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA

 5761 TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT

 5821 AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC

 5881 ATGCCATCCG TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA

 5941 TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA

 6001 CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA

 6061 AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT

 6121 TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC

 6181 GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA

 6241 TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT

 6301 TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC

 6361 TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA GGCGTATCAC GAGGCCCTTT

 6421 CGTCTCGCGC GTTTCGGTGA TGACGGTGAA AACCTCTGAC ACATGCAGCT CCCGGAGACG

 6481 GTCACAGCTT GTCTGTAAGC GGATGCCGGG AGCAGACAAG CCCGTCAGGG CGCGTCAGCG

 6541 GGTGTTGGCG GGTGTCGGGG CTGGCTTAAC TATGCGGCAT CAGAGCAGAT TGTACTGAGA

 6601 GTGCAC

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