pLVX-AmCyan1-N1 is an HIV-1-based, lentiviral expression vector that allows you to express your gene of interest fused to AmCyan1, a human codon-optimized variant of the wild-type Anemonia majano cyan fluorescent protein (1). Genes cloned into the multiple cloning site (MCS), located upstream of the AmCyan1 coding sequence, are expressed as N-terminal AmCyan1 fusion proteins. Expression of the fusion protein is driven by the constitutively

active human cytomegalovirus immediate early promoter (PCMV IE), located just upstream of the MCS. Lentiviral particles derived from the vector allow the expression of AmCyan1 fusion proteins in virtually any cell type, including primary cells. The unmodified vector expresses AmCyan1, and may be used to produce marker virus to optimize infection protocols.

pLVX-AmCyan1-N1 contains all of the viral processing elements necessary for the production of replication-incompetent lentivirus, as well as elements to improve viral titer, transgene expression, and overall vector function. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) promotes RNA processing events and enhances nuclear export of viral and transgene RNA (2), leading to increased viral titers from packaging cells, and enhanced expression of your gene of interest in target cells. In addition, the vector includes a Rev-response element (RRE), which further increases viral titers by enhancing the transport of unspliced viral RNA out of the nucleus (3). Finally, pLVX-AmCyan1-N1 also contains a central polypurine tract/central termination sequence element (cPPT/CTS). During target cell infection, this element creates a central DNA flap that increases nuclear import of the viral genome, resulting in improved vector integration and more efficient transduction (4).

In addition to lentiviral elements, pLVX-AmCyan1-N1 contains a puromycin resistance gene (Puror) under the control of the murine phosphoglycerate kinase (PGK) promoter (PPGK) for the selection of stable transductants. The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Ampr) for propagation and selection in bacteria.

載體應(yīng)用

To construct a fusion protein, the gene of interest must be cloned into pLVX-AmCyan1-N1 so that it is in-frame with the AmCyan1 coding sequence. The inserted sequence should include an initiation codon (ATG) and lack in-frame stop codons.

The fusion protein is constitutively expressed when pLVX-AmCyan1-N1 is transduced into target cells. Before the vector can be transduced, however, it must be transfected into 293T packaging cells with our Lenti-X? HT Packaging System (Cat. Nos. 632160 and 632161). This packaging system allows you to safely produce high titer, infectious, replication-incompetent, VSV-G pseudotyped lentiviral particles that can infect a wide range of cell types, including non-dividing and primary cells (5).

0001 TGGAAGGGCT AATTCACTCC CAAAGAAGAC AAGATATCCT TGATCTGTGG ATCTACCACA 
0061 CACAAGGCTA CTTCCCTGAT TAGCAGAACT ACACACCAGG GCCAGGGGTC AGATATCCAC 
0121 TGACCTTTGG ATGGTGCTAC AAGCTAGTAC CAGTTGAGCC AGATAAGGTA GAAGAGGCCA 
0181 ATAAAGGAGA GAACACCAGC TTGTTACACC CTGTGAGCCT GCATGGGATG GATGACCCGG 
0241 AGAGAGAAGT GTTAGAGTGG AGGTTTGACA GCCGCCTAGC ATTTCATCAC GTGGCCCGAG 
0301 AGCTGCATCC GGAGTACTTC AAGAACTGCT GATATCGAGC TTGCTACAAG GGACTTTCCG 
0361 CTGGGGACTT TCCAGGGAGG CGTGGCCTGG GCGGGACTGG GGAGTGGCGA GCCCTCAGAT 
0421 CCTGCATATA AGCAGCTGCT TTTTGCCTGT ACTGGGTCTC TCTGGTTAGA CCAGATCTGA 
0481 GCCTGGGAGC TCTCTGGCTA ACTAGGGAAC CCACTGCTTA AGCCTCAATA AAGCTTGCCT 
0541 TGAGTGCTTC AAGTAGTGTG TGCCCGTCTG TTGTGTGACT CTGGTAACTA GAGATCCCTC 
0601 AGACCCTTTT AGTCAGTGTG GAAAATCTCT AGCAGTGGCG CCCGAACAGG GACTTGAAAG 
0661 CGAAAGGGAA ACCAGAGGAG CTCTCTCGAC GCAGGACTCG GCTTGCTGAA GCGCGCACGG 
0721 CAAGAGGCGA GGGGCGGCGA CTGGTGAGTA CGCCAAAAAT TTTGACTAGC GGAGGCTAGA 
0781 AGGAGAGAGA TGGGTGCGAG AGCGTCAGTA TTAAGCGGGG GAGAATTAGA TCGCGATGGG 
0841 AAAAAATTCG GTTAAGGCCA GGGGGAAAGA AAAAATATAA ATTAAAACAT ATAGTATGGG 
0901 CAAGCAGGGA GCTAGAACGA TTCGCAGTTA ATCCTGGCCT GTTAGAAACA TCAGAAGGCT 
0961 GTAGACAAAT ACTGGGACAG CTACAACCAT CCCTTCAGAC AGGATCAGAA GAACTTAGAT 
1021 CATTATATAA TACAGTAGCA ACCCTCTATT GTGTGCATCA AAGGATAGAG ATAAAAGACA 
1081 CCAAGGAAGC TTTAGACAAG ATAGAGGAAG AGCAAAACAA AAGTAAGACC ACCGCACAGC 
1141 AAGCGGCCGG CCGCTGATCT TCAGACCTGG AGGAGGAGAT ATGAGGGACA ATTGGAGAAG 
1201 TGAATTATAT AAATATAAAG TAGTAAAAAT TGAACCATTA GGAGTAGCAC CCACCAAGGC 
1261 AAAGAGAAGA GTGGTGCAGA GAGAAAAAAG AGCAGTGGGA ATAGGAGCTT TGTTCCTTGG 
1321 GTTCTTGGGA GCAGCAGGAA GCACTATGGG CGCAGCGTCA ATGACGCTGA CGGTACAGGC 
1381 CAGACAATTA TTGTCTGGTA TAGTGCAGCA GCAGAACAAT TTGCTGAGGG CTATTGAGGC 
1441 GCAACAGCAT CTGTTGCAAC TCACAGTCTG GGGCATCAAG CAGCTCCAGG CAAGAATCCT 
1501 GGCTGTGGAA AGATACCTAA AGGATCAACA GCTCCTGGGG ATTTGGGGTT GCTCTGGAAA 
1561 ACTCATTTGC ACCACTGCTG TGCCTTGGAA TGCTAGTTGG AGTAATAAAT CTCTGGAACA 
1621 GATTTGGAAT CACACGACCT GGATGGAGTG GGACAGAGAA ATTAACAATT ACACAAGCTT 
1681 AATACACTCC TTAATTGAAG AATCGCAAAA CCAGCAAGAA AAGAATGAAC AAGAATTATT 
1741 GGAATTAGAT AAATGGGCAA GTTTGTGGAA TTGGTTTAAC ATAACAAATT GGCTGTGGTA 
1801 TATAAAATTA TTCATAATGA TAGTAGGAGG CTTGGTAGGT TTAAGAATAG TTTTTGCTGT 
1861 ACTTTCTATA GTGAATAGAG TTAGGCAGGG ATATTCACCA TTATCGTTTC AGACCCACCT 
1921 CCCAACCCCG AGGGGACCCG ACAGGCCCGA AGGAATAGAA GAAGAAGGTG GAGAGAGAGA 
1981 CAGAGACAGA TCCATTCGAT TAGTGAACGG ATCTCGACGG TATCGCCTTT AAAAGAAAAG 
2041 GGGGGATTGG GGGGTACAGT GCAGGGGAAA GAATAGTAGA CATAATAGCA ACAGACATAC 
2101 AAACTAAAGA ATTACAAAAA CAAATTACAA AAATTCAAAA TTTTCGGGTT TATTACAGGG 
2161 ACAGCAGAGA TCCAGTTTAT CGATAAGCTT GGGAGTTCCG CGTTACATAA CTTACGGTAA 
2221 ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG 
2281 TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT 
2341 AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG 
2401 TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC 
2461 CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC 
2521 AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA 
2581 TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA 
2641 ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA 
2701 GCAGAGCTCG TTTAGTGAAC CGTCAGATCG CCTGGAGACG CCATCCACGC TGTTTTGACC 
2761 TCCATAGAAG ACACCGACTC TACTAGAGGA TCGCTAGCGC TACCGGACTC AGATCTCGAG 
2821 CTCAAGCTTC GAATTCTGCA GTCGACGGTA CCGCGGGCCC GGGATCCACC GGTCATGGCC 
2881 CTGTCCAACA AGTTCATCGG CGACGACATG AAGATGACCT ACCACATGGA CGGCTGCGTG 
2941 AACGGCCACT ACTTCACCGT GAAGGGCGAG GGCAGCGGCA AGCCCTACGA GGGCACCCAG 
3001 ACCTCCACCT TCAAGGTGAC CATGGCCAAC GGCGGCCCCC TGGCCTTCTC CTTCGACATC 
3061 CTGTCCACCG TGTTCATGTA CGGCAACCGC TGCTTCACCG CCTACCCCAC CAGCATGCCC 
3121 GACTACTTCA AGCAGGCCTT CCCCGACGGC ATGTCCTACG AGAGAACCTT CACCTACGAG 
3181 GACGGCGGCG TGGCCACCGC CAGCTGGGAG ATCAGCCTGA AGGGCAACTG CTTCGAGCAC 
3241 AAGTCCACCT TCCACGGCGT GAACTTCCCC GCCGACGGCC CCGTGATGGC CAAGAAGACC 
3301 ACCGGCTGGG ACCCCTCCTT CGAGAAGATG ACCGTGTGCG ACGGCATCTT GAAGGGCGAC 
3361 GTGACCGCCT TCCTGATGCT GCAGGGCGGC GGCAACTACA GATGCCAGTT CCACACCTCC 
3421 TACAAGACCA AGAAGCCCGT GACCATGCCC CCCAACCACG TGGTGGAGCA CCGCATCGCC 
3481 AGAACCGACC TGGACAAGGG CGGCAACAGC GTGCAGCTGA CCGAGCACGC CGTGGCCCAC 
3541 ATCACCTCCG TGGTGCCCTT CTGAGCGGCC GCGACTCTAG ATAATTCTAC CGGGTAGGGG 
3601 AGGCGCTTTT CCCAAGGCAG TCTGGAGCAT GCGCTTTAGC AGCCCCGCTG GGCACTTGGC 
3661 GCTACACAAG TGGCCTCTGG CCTCGCACAC ATTCCACATC CACCGGTAGG CGCCAACCGG 
3721 CTCCGTTCTT TGGTGGCCCC TTCGCGCCAC CTTCTACTCC TCCCCTAGTC AGGAAGTTCC 
3781 CCCCCGCCCC GCAGCTCGCG TCGTGCAGGA CGTGACAAAT GGAAGTAGCA CGTCTCACTA 
3841 GTCTCGTGCA GATGGACAGC ACCGCTGAGC AATGGAAGCG GGTAGGCCTT TGGGGCAGCG 
3901 GCCAATAGCA GCTTTGCTCC TTCGCTTTCT GGGCTCAGAG GCTGGGAAGG GGTGGGTCCG 
3961 GGGGCGGGCT CAGGGGCGGG CTCAGGGGCG GGGCGGGCGC CCGAAGGTCC TCCGGAGGCC 
4021 CGGCATTCTG CACGCTTCAA AAGCGCACGT CTGCCGCGCT GTTCTCCTCT TCCTCATCTC 
4081 CGGGCCTTTC GACCTGCAGC CCAAGCTTAC CATGACCGAG TACAAGCCCA CGGTGCGCCT 
4141 CGCCACCCGC GACGACGTCC CCAGGGCCGT ACGCACCCTC GCCGCCGCGT TCGCCGACTA 
4201 CCCCGCCACG CGCCACACCG TCGATCCGGA CCGCCACATC GAGCGGGTCA CCGAGCTGCA 
4261 AGAACTCTTC CTCACGCGCG TCGGGCTCGA CATCGGCAAG GTGTGGGTCG CGGACGACGG 
4321 CGCCGCGGTG GCGGTCTGGA CCACGCCGGA GAGCGTCGAA GCGGGGGCGG TGTTCGCCGA 
4381 GATCGGCCCG CGCATGGCCG AGTTGAGCGG TTCCCGGCTG GCCGCGCAGC AACAGATGGA 
4441 AGGCCTCCTG GCGCCGCACC GGCCCAAGGA GCCCGCGTGG TTCCTGGCCA CCGTCGGCGT 
4501 CTCGCCCGAC CACCAGGGCA AGGGTCTGGG CAGCGCCGTC GTGCTCCCCG GAGTGGAGGC 
4561 GGCCGAGCGC GCCGGGGTGC CCGCCTTCCT GGAGACCTCC GCGCCCCGCA ACCTCCCCTT 
4621 CTACGAGCGG CTCGGCTTCA CCGTCACCGC CGACGTCGAG GTGCCCGAAG GACCGCGCAC 
4681 CTGGTGCATG ACCCGCAAGC CCGGTGCCTG ACCGCGTCTG GAACAATCAA CCTCTGGATT 
4741 ACAAAATTTG TGAAAGATTG ACTGGTATTC TTAACTATGT TGCTCCTTTT ACGCTATGTG 
4801 GATACGCTGC TTTAATGCCT TTGTATCATG CTATTGCTTC CCGTATGGCT TTCATTTTCT 
4861 CCTCCTTGTA TAAATCCTGG TTGCTGTCTC TTTATGAGGA GTTGTGGCCC GTTGTCAGGC 
4921 AACGTGGCGT GGTGTGCACT GTGTTTGCTG ACGCAACCCC CACTGGTTGG GGCATTGCCA 
4981 CCACCTGTCA GCTCCTTTCC GGGACTTTCG CTTTCCCCCT CCCTATTGCC ACGGCGGAAC 
5041 TCATCGCCGC CTGCCTTGCC CGCTGCTGGA CAGGGGCTCG GCTGTTGGGC ACTGACAATT 
5101 CCGTGGTGTT GTCGGGGAAG CTGACGTCCT TTCCATGGCT GCTCGCCTGT GTTGCCACCT 
5161 GGATTCTGCG CGGGACGTCC TTCTGCTACG TCCCTTCGGC CCTCAATCCA GCGGACCTTC 
5221 CTTCCCGCGG CCTGCTGCCG GCTCTGCGGC CTCTTCCGCG TCTTCGCCTT CGCCCTCAGA 
5281 CGAGTCGGAT CTCCCTTTGG GCCGCCTCCC CGCCTGGAAT TAATTCTGCA GTCGAGACCT 
5341 AGAAAAACAT GGAGCAATCA CAAGTAGCAA TACAGCAGCT ACCAATGCTG ATTGTGCCTG 
5401 GCTAGAAGCA CAAGAGGAGG AGGAGGTGGG TTTTCCAGTC ACACCTCAGG TACCTTTAAG 
5461 ACCAATGACT TACAAGGCAG CTGTAGATCT TAGCCACTTT TTAAAAGAAA AGAGGGGACT 
5521 GGAAGGGCTA ATTCACTCCC AACGAAGACA AGATATCCTT GATCTGTGGA TCTACCACAC 
5581 ACAAGGCTAC TTCCCTGATT AGCAGAACTA CACACCAGGG CCAGGGGTCA GATATCCACT 
5641 GACCTTTGGA TGGTGCTACA AGCTAGTACC AGTTGAGCCA GATAAGGTAG AAGAGGCCAA 
5701 TAAAGGAGAG AACACCAGCT TGTTACACCC TGTGAGCCTG CATGGGATGG ATGACCCGGA 
5761 GAGAGAAGTG TTAGAGTGGA GGTTTGACAG CCGCCTAGCA TTTCATCACG TGGCCCGAGA 
5821 GCTGCATCCG GAGTACTTCA AGAACTGCTG ATATCGAGCT TGCTACAAGG GACTTTCCGC 
5881 TGGGGACTTT CCAGGGAGGC GTGGCCTGGG CGGGACTGGG GAGTGGCGAG CCCTCAGATC 
5941 CTGCATATAA GCAGCTGCTT TTTGCCTGTA CTGGGTCTCT CTGGTTAGAC CAGATCTGAG 
6001 CCTGGGAGCT CTCTGGCTAA CTAGGGAACC CACTGCTTAA GCCTCAATAA AGCTTGCCTT 
6061 GAGTGCTTCA AGTAGTGTGT GCCCGTCTGT TGTGTGACTC TGGTAACTAG AGATCCCTCA 
6121 GACCCTTTTA GTCAGTGTGG AAAATCTCTA GCAGTAGTAG TTCATGTCAT CTTATTATTC 
6181 AGTATTTATA ACTTGCAAAG AAATGAATAT CAGAGAGTGA GAGGCCTTGA CATTGCTAGC 
6241 GTTTTACCGT CGACCTCTAG CTAGAGCTTG GCGTAATCAT GGTCATAGCT GTTTCCTGTG 
6301 TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG CCGGAAGCAT AAAGTGTAAA 
6361 GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG CGTTGCGCTC ACTGCCCGCT 
6421 TTCCAGTCGG GAAACCTGTC GTGCCAGCTG CATTAATGAA TCGGCCAACG CGCGGGGAGA 
6481 GGCGGTTTGC GTATTGGGCG CTCTTCCGCT TCCTCGCTCA CTGACTCGCT GCGCTCGGTC 
6541 GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG TAATACGGTT ATCCACAGAA 
6601 TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT 
6661 AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA 
6721 AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT 
6781 CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG 
6841 TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA GCTCACGCTG TAGGTATCTC 
6901 AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC 
6961 GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA 
7021 TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT 
7081 ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA GAAGAACAGT ATTTGGTATC 
7141 TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA 
7201 CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA 
7261 AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA 
7321 AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT 
7381 TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC 
7441 AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC 
7501 ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC 
7561 CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA 
7621 AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CAACTTTATC CGCCTCCATC 
7681 CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC 
7741 AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA 
7801 TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA 
7861 GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC AGTGTTATCA 
7921 CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT 
7981 TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT 
8041 TGCTCTTGCC CGGCGTCAAT ACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG 
8101 CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA 
8161 TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC 
8221 AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG 
8281 ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG 
8341 GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG 
8401 GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTCG ACGGATCGGG AGATCAACTT 
8461 GTTTATTGCA GCTTATAATG GTTACAAATA AAGCAATAGC ATCACAAATT TCACAAATAA 
8521 AGCATTTTTT TCACTGCATT CTAGTTGTGG TTTGTCCAAA CTCATCAATG TATCTTATCA 
8581 TGTCTGGATC AACTGGATAA CTCAAGCTAA CCAAAATCAT CCCAAACTTC CCACCCCATA 
8641 CCCTATTACC ACTGCCAATT ACCTGTGGTT TCATTTACTC TAAACCTGTG ATTCCTCTGA 
8701 ATTATTTTCA TTTTAAAGAA ATTGTATTTG TTAAATATGT ACTACAAACT TAGTAGT