出品公司: SBI
载体名称: pCDF1-MCS2-EF1-Puro
质粒类型: 慢病毒表达载体;cDNA表达载体
克隆方法: 多克隆位点,限制性内切酶
启动子: CMV
载体大小: 6606 bp
3' 测序引物及序列: EF1a-R
载体抗性: 氨苄青霉素(Ampicillin)
筛选标记: 嘌呤霉素
克隆菌株: stbl3或者 E. coli(RecA-):OmniMAX 2
宿主细胞(系): 常用细胞系(e.g.HeLa, HEK293, HT1080, H1299)
备注: pCDF1-MCS2-EF1-Puro慢病毒表达载体是基于FIV的慢病毒载体;
用于cDNA表达和克隆;高效转染细胞,建立稳定细胞系 ,表达水平高;
产品目录号: CD110B-1
稳定性: 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 慢病毒(FIV)



pCDF1-MCS2-EF1-Puro 载体特征


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. 


LOCUS       pCDF1-MCS2-EF1-Puro	6606 bp 	DNA	SYN
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     source          1..6606
                     /mol_type="other DNA"
     misc_feature    287..307
     misc_feature    1165..1180
     misc_feature    1633..1653
     promoter        1634..1703
     misc_feature    1677..1696
     misc_feature    1679..1703
     gene            2358..2957
                     /label="puro (variant)"
                     /gene="puro (variant)"
     CDS             2358..2957
                     /label="ORF frame 3"
     misc_feature    2966..3541
     CDS             3054..3566
                     /label="ORF frame 3"
     CDS             3067..3639
                     /label="ORF frame 1"
     misc_feature    3653..3678
     misc_feature    3655..3678
     terminator      3893..4012
     misc_feature    3981..4000
     rep_origin      4030..4107
     misc_feature    4092..4111
     promoter        complement(4199..4217)
     misc_feature    complement(4216..4238)
     promoter        complement(4252..4281)
     gene            complement(5364..6224)
     CDS             complement(5364..6224)
                     /label="ORF frame 2"
     promoter        complement(6266..6294)
     misc_feature    complement(6453..6475)
    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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