pRetroX-IRES-ZsGreen1

pRetroX-IRES-ZsGreen1 is a bicistronic, fl uorescent, retroviral vector that allows both a gene of interest and the ZsGreen1 gene to be translated from a single bicistronic mRNA. pRetroXIRES- ZsGreen1 is designed for effi cient delivery and selection (by fl ow cytometry or other methods) of stably transduced mammalian cells expressing the ZsGreen1 fl uorescent protein and the protein of interest. This vector can be used to obtain stable cell lines without timeconsuming drug and clonal selection. ZsGreen1 is derived from the reef coral Zoanthus sp. green fl uorescent protein (ZsGreen) and is easily detected with standard FITC fi lter sets (1).

Bicistronic expression from this vector is facilitated by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). This IRES facilitates cap-independent translation from an internal start site at the IRES/ZsGreen1 junction (2). ZsGreen1 is a human codon optimized ZsGreen variant that encodes the brightest commercially available green fl uorescent protein (1). This retroviral vector is derived from the pMIN series of vectors (3, 4). These optimized vectors have the ability to produce high viral titers, express genes at high levels, and, due to the absence of retroviral coding sequences, exhibit improved safety profi les. The multiple cloning site (MCS) in pRetroX-IRES-ZsGreen1 is between the 5’ MMLV LTR and the IRES sequence. Genes cloned into the MCS are expressed as a bicistronic message transcribed from the 5’ LTR.

pRetroX-IRES-ZsGreen1 contains all of the necessary viral RNA processing elements; these include the 5’ and 3’ LTRs, the packaging signal (ψ), and the tRNA primer-binding site. For safety reasons, however, the vector lacks the structural genes (gag, pol, and env) necessary for retroviral particle formation and replication. pRetroX-IRES-ZsGreen1 contains a ColE1 origin of replication, and an E. coli Ampr gene for propagation and selection in bacteria.

载体应用

pRetroX-IRES-ZsGreen1 is designed to effi ciently deliver and co-express your gene of interest and ZsGreen1 in any mitotically-active mammalian cell. When cloning into pRetroX-IRES-ZsGreen1, your gene of interest should contain an initiation codon (ATG) and a stop codon. The vector can be infected or transfected into mammalian cells. If required, stable transformants can be selected by fl ow cytometry or limiting dilution.

In order to infect mammalian cells with pRetroX-IRES-ZsGreen1, the vector must be transfected into a packaging cell line, such as the RetroPack? PT67 Cell line (631510), AmphoPack?-293 (631505), EcoPack?2- 293 (631507), Pantropic Expression System (631512), or Retro-X? Universal Packaging System (631530). These cell lines package RNA from the vector into infectious, replication-incompetent, retroviral particles. Such retroviral particles can infect target cells and transmit the gene of interest, but cannot replicate within these cells due to the absence of viral structural genes. The separate introduction and integration of the structural genes into the packaging cell line minimizes the chance of producing replication-competent virus due to recombination events during cell proliferation.

0001 TGAAAGACCC CACCTGTAGG TTTGGCAAGC TAGCTTAAGT AACGCCATTT TGCAAGGCAT 
0061 GGAAAAATAC ATAACTGAGA ATAGAAAAGT TCAGATCAAG GTCAGGAACA GATGGAACAG 
0121 CTGAATATGG GCCAAACAGG ATATCTGTGG TAAGCAGTTC CTGCCCCGGC TCAGGGCCAA 
0181 GAACAGATGG AACAGCTGAA TATGGGCCAA ACAGGATATC TGTGGTAAGC AGTTCCTGCC 
0241 CCGGCTCAGG GCCAAGAACA GATGGTCCCC AGATGCGGTC CAGCCCTCAG CAGTTTCTAG 
0301 AGAACCATCA GATGTTTCCA GGGTGCCCCA AGGACCTGAA ATGACCCTGT GCCTTATTTG 
0361 AACTAACCAA TCAGTTCGCT TCTCGCTTCT GTTCGCGCGC TTCTGCTCCC CGGGCTCAAT 
0421 AAAAGAGCCC ACAACCCCTC ACTCGGCGCG CCAGTCCTCC GATTGACTGA GTCGCCCGGG 
0481 TACCCGTGTA TCCAATAAAC CCTCTTGCAG TTGCATCCGA CTTGTGGCCT CGCTGTTCCT 
0541 TGGGAGGGTC TCCTCTGAGT GATTGACTAC CCGTCAGCGG GGGTCTTTCA TTTGGGGGCT 
0601 CGTCCGGGAT CGGGAGACCC CTGCCCAGGG ACCACCGACC CACCACCGGG AGGTAAGCTG 
0661 GCCAGCAACT TATCTGTGTC TGTCCGATTG TCTAGTGTCT ATGACTGATT TTATGCGCCT 
0721 GCGTCGGTAC TAGTTAGCTA ACTAGCTCTG TATCTGGCGG ACCCGTGGTG GAACTGACGA 
0781 GTTCGGAACA CCCGGCCGCA ACCCTGGGAG ACGTCCCAGG GACTTCGGGG GCCGTTTTTG 
0841 TGGCCCGACC TGAGTCCAAA AATCCCGATC GTTTTGGACT CTTTGGTGCA CCCCCCTTAG 
0901 AGGAGGGATA TGTGGTTCTG GTAGGAGACG AGAACCTAAA ACAGTTCCCG CCTCCGTCTG 
0961 AATTTTTGCT TTCGGTTTGG GACCGAAGCC GCGCCGCGCG TCTTGTCTGC TGCAGCATCG 
1021 TTCTGTGTTG TCTCTGTCTG ACTGTGTTTC TGTATTTGTC TGAAAATACG CGTGTCGAGC 
1081 TTTTGGAGTA CGTCGTCTTT AGGTTGGGGG GAGGGGTTTT ATGCGATGGA GTTTCCCCAC 
1141 ACTGAGTGGG TGGAGACTGA AGTTAGGCCA GCTTGGCACT TGATGTAATT CTCCTTGGAA 
1201 TTTGCCCTTT TTGAGTTTGG ATCTTGGTTC ATTCTCAAGC CTCAGACAGT GGTTCAAAGT 
1261 TTTTTTCTTC CATTTCAGCC GTCGTGAAAA CTACCCCTAA AAGCCAAAAA AATCGAATTC 
1321 CCAAACTTAA GCTTGGTACC GAGCTCGGAT CTCACGTGGG CCCGCGGCCG CAGATCTATC 
1381 GATGGATCCG TGCCCCTCTC CCTCCCCCCC CCCTAACGTT ACTGGCCGAA GCCGCTTGGA 
1441 ATAAGGCCGG TGTGCGTTTG TCTATATGTT ATTTTCCACC ATATTGCCGT CTTTTGGCAA 
1501 TGTGAGGGCC CGGAAACCTG GCCCTGTCTT CTTGACGAGC ATTCCTAGGG GTCTTTCCCC 
1561 TCTCGCCAAA GGAATGCAAG GTCTGTTGAA TGTCGTGAAG GAAGCAGTTC CTCTGGAAGC 
1621 TTCTTGAAGA CAAACAACGT CTGTAGCGAC CCTTTGCAGG CAGCGGAACC CCCCACCTGG 
1681 CGACAGGTGC CTCTGCGGCC AAAAGCCACG TGTATAAGAT ACACCTGCAA AGGCGGCACA 
1741 ACCCCAGTGC CACGTTGTGA GTTGGATAGT TGTGGAAAGA GTCAAATGGC TCACCTCAAG 
1801 CGTATTCAAC AAGGGGCTGA AGGATGCCCA GAAGGTACCC CATTGTATGG GATCTGATCT 
1861 GGGGCCTCGG TGCACATGCT TTACATGTGT TTAGTCGAGG TTAAAAAACG TCTAGGCCCC 
1921 CCGAACCACG GGGACGTGGT TTTCCTTTGA AAAACACGAT GATAATATGG CCCAGTCCAA 
1981 GCACGGCCTG ACCAAGGAGA TGACCATGAA GTACCGCATG GAGGGCTGCG TGGACGGCCA 
2041 CAAGTTCGTG ATCACCGGCG AGGGCATCGG CTACCCCTTC AAGGGCAAGC AGGCCATCAA 
2101 CCTGTGCGTG GTGGAGGGCG GCCCCTTGCC CTTCGCCGAG GACATCTTGT CCGCCGCCTT 
2161 CATGTACGGC AACCGCGTGT TCACCGAGTA CCCCCAGGAC ATCGTCGACT ACTTCAAGAA 
2221 CTCCTGCCCC GCCGGCTACA CCTGGGACCG CTCCTTCCTG TTCGAGGACG GCGCCGTGTG 
2281 CATCTGCAAC GCCGACATCA CCGTGAGCGT GGAGGAGAAC TGCATGTACC ACGAGTCCAA 
2341 GTTCTACGGC GTGAACTTCC CCGCCGACGG CCCCGTGATG AAGAAGATGA CCGACAACTG 
2401 GGAGCCCTCC TGCGAGAAGA TCATCCCCGT GCCCAAGCAG GGCATCTTGA AGGGCGACGT 
2461 GAGCATGTAC CTGCTGCTGA AGGACGGTGG CCGCTTGCGC TGCCAGTTCG ACACCGTGTA 
2521 CAAGGCCAAG TCCGTGCCCC GCAAGATGCC CGACTGGCAC TTCATCCAGC ACAAGCTGAC 
2581 CCGCGAGGAC CGCAGCGACG CCAAGAACCA GAAGTGGCAC CTGACCGAGC ACGCCATCGC 
2641 CTCCGGCTCC GCCTTGCCCT GAGCGGCTTC GAGGATAAAA TAAAAGATTT TATTTAGTCT 
2701 CCAGAAAAAG GGGGGAATGA AAGACCCCAC CTGTAGGTTT GGCAAGCTAG CTTAAGTAAC 
2761 GCCATTTTGC AAGGCATGGA AAAATACATA ACTGAGAATA GAGAAGTTCA GATCAAGGTC 
2821 AGGAACAGAT GGAACAGCTG AATATGGGCC AAACAGGATA TCTGTGGTAA GCAGTTCCTG 
2881 CCCCGGCTCA GGGCCAAGAA CAGATGGAAC AGCTGAATAT GGGCCAAACA GGATATCTGT 
2941 GGTAAGCAGT TCCTGCCCCG GCTCAGGGCC AAGAACAGAT GGTCCCCAGA TGCGGTCCAG 
3001 CCCTCAGCAG TTTCTAGAGA ACCATCAGAT GTTTCCAGGG TGCCCCAAGG ACCTGAAATG 
3061 ACCCTGTGCC TTATTTGAAC TAACCAATCA GTTCGCTTCT CGCTTCTGTT CGCGCGCTTC 
3121 TGCTCCCCGA GCTCAATAAA AGAGCCCACA ACCCCTCACT CGGGGCGCCA GTCCTCCGAT 
3181 TGACTGAGTC GCCCGGGTAC CCGTGTATCC AATAAACCCT CTTGCAGTTG CATCCGACTT 
3241 GTGGTCTCGC TGTTCCTTGG GAGGGTCTCC TCTGAGTGAT TGACTACCCG TCAGCGGGGG 
3301 TCTTTCATTG AATTCGTAAT CATGGTCATA GCTGTTTCCT GTGTGAAATT GTTATCCGCT 
3361 CACAATTCCA CACAACATAC GAGCCGGAAG CATAAAGTGT AAAGCCTGGG GTGCCTAATG 
3421 AGTGAGCTAA CTCACATTAA TTGCGTTGCG CTCACTGCCC GCTTTCCAGT CGGGAAACCT 
3481 GTCGTGCCAG CTGCATTAAT GAATCGGCCA ACGCGCGGGG AGAGGCGGTT TGCGTATTGG 
3541 GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC 
3601 GGTATCAGCT CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG 
3661 AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT 
3721 GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA 
3781 GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT 
3841 CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC 
3901 GGGAAGCGTG GCGCTTTCTC ATAGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT 
3961 TCGCTCCAAG CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC 
4021 CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC 
4081 CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG 
4141 GTGGCCTAAC TACGGCTACA CTAGAAGAAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC 
4201 AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG 
4261 CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA 
4321 TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT 
4381 TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG 
4441 TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT 
4501 CAGTGAGGCA CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTGACTCCC 
4561 CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT 
4621 ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG 
4681 GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAATTGTTG 
4741 CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG CGCAACGTTG TTGCCATTGC 
4801 TACAGGCATC GTGGTGTCAC GCTCGTCGTT TGGTATGGCT TCATTCAGCT CCGGTTCCCA 
4861 ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG 
4921 TCCTCCGATC GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC 
4981 ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC TTTTCTGTGA CTGGTGAGTA 
5041 CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG AGTTGCTCTT GCCCGGCGTC 
5101 AATACGGGAT AATACCGCGC CACATAGCAG AACTTTAAAA GTGCTCATCA TTGGAAAACG 
5161 TTCTTCGGGG CGAAAACTCT CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC 
5221 CACTCGTGCA CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC 
5281 AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGA AATGTTGAAT 
5341 ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT CAGGGTTATT GTCTCATGAG 
5401 CGGATACATA TTTGAATGTA TTTAGAAAAA TAAACAAATA GGGGTTCCGC GCACATTTCC 
5461 CCGAAAAGTG CCACCTGACG TCTAAGAAAC CATTATTATC ATGACATTAA CCTATAAAAA 
5521 TAGGCGTATC ACGAGGCCCT TTCGTCTCGC GCGTTTCGGT GATGACGGTG AAAACCTCTG 
5581 ACACATGCAG CTCCCGGAGA CGGTCACAGC TTGTCTGTAA GCGGATGCCG GGAGCAGACA 
5641 AGCCCGTCAG GGCGCGTCAG CGGGTGTTGG CGGGTGTCGG GGCTGGCTTA ACTATGCGGC 
5701 ATCAGAGCAG ATTGTACTGA GAGTGCACCA TATGCGGTGT GAAATACCGC ACAGATGCGT 
5761 AAGGAGAAAA TACCGCATCA GGCGCCATTC GCCATTCAGG CTGCGCAACT GTTGGGAAGG 
5821 GCGATCGGTG CGGGCCTCTT CGCTATTACG CCAGCTGGCG AAAGGGGGAT GTGCTGCAAG 
5881 GCGATTAAGT TGGGTAACGC CAGGGTTTTC CCAGTCACGA CGTTGTAAAA CGACGGCCAG 
5941 TGCCAAGCTT ATG