pCDH-CMV-MCS-EF1-copGFP-T2A-Puro

pCDH-CMV-MCS-EF1-copGFP-T2A-Puro

pCDH-CMV-MCS-EF1-copGFP-T2A-Puro载体基本信息：

pCDH-CMV-MCS-EF1-copGFP-T2A-Puro载体质粒图谱和多克隆位点信息：

pCDH-CMV-MCS-EF1-copGFP-T2A-Puro载体简介：

背景简介：

This manual provides details and information necessary to generate expression constructs of your gene of interest in the pCDH cDNA Cloning and Expression Lentivectors. Specifically, it provides critical instructions on amplification and cloning cDNA into the pCDH vectors, and verification of the final expression constructs. This manual does not include information on packaging the pCDH expression constructs into pseudotyped viral particles or transducing your target cells of choice with these particles. This information is available in the user manual Lentivector Expression Systems: Guide to Packaging and Transduction of Target Cells which is available on the SBI website. Before using the reagents and material supplied with this system, please read the entire manual.

基于HIV-1的pCDH 慢病毒载体特征：

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

 WPRE element—enhances stability and translation of the CMV-driven transcripts.

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

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

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

 SV40 origin—for stable propagation of the pCDH 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.

pCDH 慢病毒表达载体的优势：

Lentiviral expression vectors are the most effective vehicles for the delivery 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-to-medium 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.

pCDH 慢病毒载体的包装载体及细胞系

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 co-transfected 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 System user manual.

启动子的选择：

SBI provides a collection of cDNA cloning and expression vectors for various applications. A gene of interest can be cloned under a CMV or EF1 promoter with or without another expression cassette for a reporter gene (copGFP or PuroR). Genes can be either expressed transiently through transfection or stably expressed in a target cell line through transduction with packaged viral particles.

The major concern of cDNA expression in lentivectors is the efficiency level and stability of expression in target cell lines.

The Cytomegalovirus (CMV) promoter is a strong and most commonly used viral promoter that constitutively expresses downstream genes. While the CMV promoter works perfectly in the most common cell lines, it shows poor expression in some stem cell lines and hematopoietic cell lines (R.F. Doll, 1996; E.D. Papadakis, 2004).

The housekeeping elongation factor 1α (EF1) promoter has been shown to exceed and outlast CMV-mediated expression in retroviral, lentiviral, and adenoviral vectors, in hematopoietic cell lines (K. Tokushige 1997; H. Nakai, 1998; C. Teschendorf, 2002). EF1 also performs well in most common cell lines.

MSCV promoter is the 5’-LTR promoter of murine stem cell virus. When a portion of the U3 region of the 3’ HIV LTR was replaced with the U3 region of MSCV LTR, the resulted hybrid HIV/MSCV LTR has dramatically increased the transgene expression level in human CD34+ hematopoietic cells (J.K. Choi, 2001). After integration into genomic DNA, this promoter transcribes a long transcript with an intron in the 5’UTR flanked with splice donor and acceptor sites derived from the lentiviral vector. Further studies found that additional CpG mutations in the MSCV LTR reduced transcriptional silencing in embryonic stem cells (C.S. Swindle, 2004). We constructed cDNA expression vectors with the CpG-deficient MSCV incorporated into the 3’ HIV LTR. After integration into genomic DNA, 3’MSCV/LTR will replace the 5’LTR and provide a high level of expression of the target gene and reporter gene downstream.

SBI第三代慢病毒载体

SBI offers a third generation of the most popular HIV-1 based lentivector expression system which consists of three maincomponents:

(1) The lentiviral expression vector (e.g., pCDH-EF1-MCS-T2A-Puro)

(2) The lentiviral packaging plasmids (e.g., pPACKH1 Packaging Plasmid mix)

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

2A Peptide-enabled dual expression system

Coexpression of a reporter gene together with a gene of interest is a useful approach for selecting transfected or transduced cells. This is commonly achieved by using two independent internal promoters, such as CMV and EF1 in pCDH-CMV-MCSEF1- copGFP, or by linking two transgenes with an internal ribosomal entry site (IRES) element in a single bicistronic transcript. Many dual promoter pairs have shown a high level of expression of both transgenes in standard cell lines— however, promoter interference often occurs in some cell lines. There are also two main problems that limit the use of IRES: the large size and the imbalanced expression between the first and second cistrons (H. Mizuguchi, 2000; X.Yu, 2003).

The “self-cleaving” 2A peptides have been used successfully to generate multiple proteins from a single promoter in many applications (P. de Felipe, 2004; M.J. Osborn, 2005; P. de Felipe, 2006). The 2A-like sequences exist in several  viruses  and are used to mediate protein cleavage from a single open reading frame. Through a ribosomal skip mechanism, the 2A peptide prevents normal peptide bond formation between the 2A glycine and the 2B proline without affecting the translation of 2B (M.L. Donnelly, 2001):SBI’s cDNA expression vectors incorporate the 2A-like sequence (T2A) from the insect virus Thosea asigna to mediate the coexpression of a reporter gene with the target cDNA. Reporter genes have been cloned at either the first or second positions, and we achieved high expression levels at both locations.

pCDH-CMV-MCS-EF1-copGFP-T2A-Puro载体序列：

ORIGIN

        1 ACGCGTGTAG TCTTATGCAA TACTCTTGTA GTCTTGCAAC ATGGTAACGA TGAGTTAGCA

       61 ACATGCCTTA CAAGGAGAGA AAAAGCACCG TGCATGCCGA TTGGTGGAAG TAAGGTGGTA

      121 CGATCGTGCC TTATTAGGAA GGCAACAGAC GGGTCTGACA TGGATTGGAC GAACCACTGA

      181 ATTGCCGCAT TGCAGAGATA TTGTATTTAA GTGCCTAGCT CGATACAATA AACGGGTCTC

      241 TCTGGTTAGA CCAGATCTGA GCCTGGGAGC TCTCTGGCTA ACTAGGGAAC CCACTGCTTA

      301 AGCCTCAATA AAGCTTGCCT TGAGTGCTTC AAGTAGTGTG TGCCCGTCTG TTGTGTGACT

      361 CTGGTAACTA GAGATCCCTC AGACCCTTTT AGTCAGTGTG GAAAATCTCT AGCAGTGGCG

      421 CCCGAACAGG GACCTGAAAG CGAAAGGGAA ACCAGAGCTC TCTCGACGCA GGACTCGGCT

      481 TGCTGAAGCG CGCACGGCAA GAGGCGAGGG GCGGCGACTG GTGAGTACGC CAAAAATTTT

      541 GACTAGCGGA GGCTAGAAGG AGAGAGATGG GTGCGAGAGC GTCAGTATTA AGCGGGGGAG

      601 AATTAGATCG CGATGGGAAA AAATTCGGTT AAGGCCAGGG GGAAAGAAAA AATATAAATT

      661 AAAACATATA GTATGGGCAA GCAGGGAGCT AGAACGATTC GCAGTTAATC CTGGCCTGTT

      721 AGAAACATCA GAAGGCTGTA GACAAATACT GGGACAGCTA CAACCATCCC TTCAGACAGG

      781 ATCAGAAGAA CTTAGATCAT TATATAATAC AGTAGCAACC CTCTATTGTG TGCATCAAAG

      841 GATAGAGATA AAAGACACCA AGGAAGCTTT AGACAAGATA GAGGAAGAGC AAAACAAAAG

      901 TAAGACCACC GCACAGCAAG CGGCCACTGA TCTTCAGACC TGGAGGAGGA GATATGAGGG

      961 ACAATTGGAG AAGTGAATTA TATAAATATA AAGTAGTAAA AATTGAACCA TTAGGAGTAG

     1021 CACCCACCAA GGCAAAGAGA AGAGTGGTGC AGAGAGAAAA AAGAGCAGTG GGAATAGGAG

     1081 CTTTGTTCCT TGGGTTCTTG GGAGCAGCAG GAAGCACTAT GGGCGCAGCC TCAATGACGC

     1141 TGACGGTACA GGCCAGACAA TTATTGTCTG GTATAGTGCA GCAGCAGAAC AATTTGCTGA

     1201 GGGCTATTGA GGCGCAACAG CATCTGTTGC AACTCACAGT CTGGGGCATC AAGCAGCTCC

     1261 AGGCAAGAAT CCTGGCTGTG GAAAGATACC TAAAGGATCA ACAGCTCCTG GGGATTTGGG

     1321 GTTGCTCTGG AAAACTCATT TGCACCACTG CTGTGCCTTG GAATGCTAGT TGGAGTAATA

     1381 AATCTCTGGA ACAGATTGGA ATCACACGAC CTGGATGGAG TGGGACAGAG AAATTAACAA

     1441 TTACACAAGC TTAATACACT CCTTAATTGA AGAATCGCAA AACCAGCAAG AAAAGAATGA

     1501 ACAAGAATTA TTGGAATTAG ATAAATGGGC AAGTTTGTGG AATTGGTTTA ACATAACAAA

     1561 TTGGCTGTGG TATATAAAAT TATTCATAAT GATAGTAGGA GGCTTGGTAG GTTTAAGAAT

     1621 AGTTTTTGCT GTACTTTCTA TAGTGAATAG AGTTAGGCAG GGATATTCAC CATTATCGTT

     1681 TCAGACCCAC CTCCCAACCC CGAGGGGACC CGACAGGCCC GAAGGAATAG AAGAAGAAGG

     1741 TGGAGAGAGA GACAGAGACA GATCCATTCG ATTAGTGAAC GGATCTCGAC GGTATCGGTT

     1801 AACTTTTAAA AGAAAAGGGG GGATTGGGGG GTACAGTGCA GGGGAAAGAA TAGTAGACAT

     1861 AATAGCAACA GACATACAAA CTAAAGAATT ACAAAAACAA ATTACAAAAT TCAAAATTTT

     1921 ATCGATACTA GTATTATGCC CAGTACATGA CCTTATGGGA CTTTCCTACT TGGCAGTACA

     1981 TCTACGTATT AGTCATCGCT ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC

     2041 GTGGATAGCG GTTTGACTCA CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA

     2101 GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG TCGTAACAAC TCCGCCCCAT

     2161 TGACGCAAAT GGGCGGTAGG CGTGTACGGT GGGAGGTTTA TATAAGCAGA GCTCGTTTAG

     2221 TGAACCGTCA GATCGCCTGG AGACGCCATC CACGCTGTTT TGACCTCCAT AGAAGATTCT

     2281 AGAGCTAGCG AATTCGAATT TAAATCGGAT CCGCGGCCGC GAAGGATCTG CGATCGCTCC

     2341 GGTGCCCGTC AGTGGGCAGA GCGCACATCG CCCACAGTCC CCGAGAAGTT GGGGGGAGGG

     2401 GTCGGCAATT GAACGGGTGC CTAGAGAAGG TGGCGCGGGG TAAACTGGGA AAGTGATGTC

     2461 GTGTACTGGC TCCGCCTTTT TCCCGAGGGT GGGGGAGAAC CGTATATAAG TGCAGTAGTC

     2521 GCCGTGAACG TTCTTTTTCG CAACGGGTTT GCCGCCAGAA CACAGCTGAA GCTTCGAGGG

     2581 GCTCGCATCT CTCCTTCACG CGCCCGCCGC CCTACCTGAG GCCGCCATCC ACGCCGGTTG

     2641 AGTCGCGTTC TGCCGCCTCC CGCCTGTGGT GCCTCCTGAA CTGCGTCCGC CGTCTAGGTA

     2701 AGTTTAAAGC TCAGGTCGAG ACCGGGCCTT TGTCCGGCGC TCCCTTGGAG CCTACCTAGA

     2761 CTCAGCCGGC TCTCCACGCT TTGCCTGACC CTGCTTGCTC AACTCTACGT CTTTGTTTCG

     2821 TTTTCTGTTC TGCGCCGTTA CAGATCCAAG CTGTGACCGG CGCCTACGCT AGATGGAGAG

     2881 CGACGAGAGC GGCCTGCCCG CCATGGAGAT CGAGTGCCGC ATCACCGGCA CCCTGAACGG

     2941 CGTGGAGTTC GAGCTGGTGG GCGGCGGAGA GGGCACCCCC AAGCAGGGCC GCATGACCAA

     3001 CAAGATGAAG AGCACCAAAG GCGCCCTGAC CTTCAGCCCC TACCTGCTGA GCCACGTGAT

     3061 GGGCTACGGC TTCTACCACT TCGGCACCTA CCCCAGCGGC TACGAGAACC CCTTCCTGCA

     3121 CGCCATCAAC AACGGCGGCT ACACCAACAC CCGCATCGAG AAGTACGAGG ACGGCGGCGT

     3181 GCTGCACGTG AGCTTCAGCT ACCGCTACGA GGCCGGCCGC GTGATCGGCG ACTTCAAGGT

     3241 GGTGGGCACC GGCTTCCCCG AGGACAGCGT GATCTTCACC GACAAGATCA TCCGCAGCAA

     3301 CGCCACCGTG GAGCACCTGC ACCCCATGGG CGATAACGTG CTGGTGGGCA GCTTCGCCCG

     3361 CACCTTCAGC CTGCGCGACG GCGGCTACTA CAGCTTCGTG GTGGACAGCC ACATGCACTT

     3421 CAAGAGCGCC ATCCACCCCA GCATCCTGCA GAACGGGGGC CCCATGTTCG CCTTCCGCCG

     3481 CGTGGAGGAG CTGCACAGCA ACACCGAGCT GGGCATCGTG GAGTACCAGC ACGCCTTCAA

     3541 GACCCCCATC GCCTTCGCCA GATCCCGCGC TCAGTCGTCC AATTCTGCCG TGGACGGCAC

     3601 CGCCGGACCC GGCTCCACCG GATCTCGCTG AGGGCAGAGG AAGTCTTCTA ACATGCGGTG

     3661 ACGTGGAGGA GAATCCCGGC CCTTCCGGGA TGACCGAGTA CAAGCCCACG GTGCGCCTCG

     3721 CCACCCGCGA CGACGTCCCC AGGGCCGTAC GCACCCTCGC CGCCGCGTTC GCCGACTACC

     3781 CCGCCACGCG CCACACCGTC GATCCGGACC GCCACATCGA GCGGGTCACC GAGCTGCAAG

     3841 AACTCTTCCT CACGCGCGTC GGGCTCGACA TCGGCAAGGT GTGGGTCGCG GACGACGGCG

     3901 CCGCGGTGGC GGTCTGGACC ACGCCGGAGA GCGTCGAAGC GGGGGCGGTG TTCGCCGAGA

     3961 TCGGCCCGCG CATGGCCGAG TTGAGCGGTT CCCGGCTGGC CGCGCAGCAA CAGATGGAAG

     4021 GCCTCCTGGC GCCGCACCGG CCCAAGGAGC CCGCGTGGTT CCTGGCCACC GTCGGCGTCT

     4081 CGCCCGACCA CCAGGGCAAG GGTCTGGGCA GCGCCGTCGT GCTCCCCGGA GTGGAGGCGG

     4141 CCGAGCGCGC CGGGGTGCCC GCCTTCCTGG AGACCTCCGC GCCCCGCAAC CTCCCCTTCT

     4201 ACGAGCGGCT CGGCTTCACC GTCACCGCCG ACGTCGAGGT GCCCGAAGGA CCGCGCACCT

     4261 GGTGCATGAC CCGCAAGCCC GGTGCCTGAG TCGACAATCA ACCTCTGGAT TACAAAATTT

     4321 GTGAAAGATT GACTGGTATT CTTAACTATG TTGCTCCTTT TACGCTATGT GGATACGCTG

     4381 CTTTAATGCC TTTGTATCAT GCTATTGCTT CCCGTATGGC TTTCATTTTC TCCTCCTTGT

     4441 ATAAATCCTG GTTGCTGTCT CTTTATGAGG AGTTGTGGCC CGTTGTCAGG CAACGTGGCG

     4501 TGGTGTGCAC TGTGTTTGCT GACGCAACCC CCACTGGTTG GGGCATTGCC ACCACCTGTC

     4561 AGCTCCTTTC CGGGACTTTC GCTTTCCCCC TCCCTATTGC CACGGCGGAA CTCATCGCCG

     4621 CCTGCCTTGC CCGCTGCTGG ACAGGGGCTC GGCTGTTGGG CACTGACAAT TCCGTGGTGT

     4681 TGTCGGGGAA ATCATCGTCC TTTCCTTGGC TGCTCGCCTG TGTTGCCACC TGGATTCTGC

     4741 GCGGGACGTC CTTCTGCTAC GTCCCTTCGG CCCTCAATCC AGCGGACCTT CCTTCCCGCG

     4801 GCCTGCTGCC GGCTCTGCGG CCTCTTCCGC GTCTTCGCCT TCGCCCTCAG ACGAGTCGGA

     4861 TCTCCCTTTG GGCCGCCTCC CCGCCTGGTA CCTTTAAGAC CAATGACTTA CAAGGCAGCT

     4921 GTAGATCTTA GCCACTTTTT AAAAGAAAAG GGGGGACTGG AAGGGCTAAT TCACTCCCAA

     4981 CGAAAATAAG ATCTGCTTTT TGCTTGTACT GGGTCTCTCT GGTTAGACCA GATCTGAGCC

     5041 TGGGAGCTCT CTGGCTAACT AGGGAACCCA CTGCTTAAGC CTCAATAAAG CTTGCCTTGA

     5101 GTGCTTCAAG TAGTGTGTGC CCGTCTGTTG TGTGACTCTG GTAACTAGAG ATCCCTCAGA

     5161 CCCTTTTAGT CAGTGTGGAA AATCTCTAGC AGTAGTAGTT CATGTCATCT TATTATTCAG

     5221 TATTTATAAC TTGCAAAGAA ATGAATATCA GAGAGTGAGA GGAACTTGTT TATTGCAGCT

     5281 TATAATGGTT ACAAATAAAG CAATAGCATC ACAAATTTCA CAAATAAAGC ATTTTTTTCA

     5341 CTGCATTCTA GTTGTGGTTT GTCCAAACTC ATCAATGTAT CTTATCATGT CTGGCTCTAG

     5401 CTATCCCGCC CCTAACTCCG CCCAGTTCCG CCCATTCTCC GCCCCATGGC TGACTAATTT

     5461 TTTTTATTTA TGCAGAGGCC GAGGCCGCCT CGGCCTCTGA GCTATTCCAG AAGTAGTGAG

     5521 GAGGCTTTTT TGGAGGCCTA GACTTTTGCA GAGACGGCCC AAATTCGTAA TCATGGTCAT

     5581 AGCTGTTTCC TGTGTGAAAT TGTTATCCGC TCACAATTCC ACACAACATA CGAGCCGGAA

     5641 GCATAAAGTG TAAAGCCTGG GGTGCCTAAT GAGTGAGCTA ACTCACATTA ATTGCGTTGC

     5701 GCTCACTGCC CGCTTTCCAG TCGGGAAACC TGTCGTGCCA GCTGCATTAA TGAATCGGCC

     5761 AACGCGCGGG GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACT

     5821 CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC

     5881 GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAA

     5941 AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG

     6001 ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA

     6061 GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC

     6121 TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC

     6181 GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC

     6241 CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG

     6301 TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT

     6361 ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGGA

     6421 CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT

     6481 CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA

     6541 TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG

     6601 CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT

     6661 TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT

     6721 AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC

     6781 TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG

     6841 GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA CCCACGCTCA CCGGCTCCAG

     6901 ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT

     6961 TATCCGCCTC CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG

     7021 TTAATAGTTT GCGCAACGTT GTTGCCATTG CTACAGGCAT CGTGGTGTCA CGCTCGTCGT

     7081 TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG GCGAGTTACA TGATCCCCCA

     7141 TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG

     7201 CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT

     7261 CCGTAAGATG CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA

     7321 TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA TAATACCGCG CCACATAGCA

     7381 GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT

     7441 TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT

     7501 CTTTTACTTT CACCAGCGTT TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA

     7561 AGGGAATAAG GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT

     7621 GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA

     7681 ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC GTCTAAGAAA

     7741 CCATTATTAT CATGACATTA ACCTATAAAA ATAGGCGTAT CACGAGGCCC TTTCGTCTCG

     7801 CGCGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA GCTCCCGGAG ACGGTCACAG

     7861 CTTGTCTGTA AGCGGATGCC GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA GCGGGTGTTG

     7921 GCGGGTGTCG GGGCTGGCTT AACTATGCGG CATCAGAGCA GATTGTACTG AGAGTGCACC

     7981 ATATGCGGTG TGAAATACCG CACAGATGCG TAAGGAGAAA ATACCGCATC AGGCGCCATT

     8041 CGCCATTCAG GCTGCGCAAC TGTTGGGAAG GGCGATCGGT GCGGGCCTCT TCGCTATTAC

     8101 GCCAGCTGGC GAAAGGGGGA TGTGCTGCAA GGCGATTAAG TTGGGTAACG CCAGGGTTTT

     8161 CCCAGTCACG ACGTTGTAAA ACGACGGCCA GTGCCAAGCT G

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pCDH-CMV-MCS-EF1-copGFP-T2A-Puro其他相关慢病毒载体：