pRetroQ-mCherry-C1

pRetroQ-mCherry-C1

pRetroQ-mCherry-C1逆病毒载体基本信息：

pRetroQ-mCherry-C1载体质粒图谱和多克隆位点信息：

pRetroQ-mCherry-C1载体简介：

pRetroQ-mCherry-C1载体描述

pRetroQ-mCherry-C1 is a high-titer, self-inactivating retroviral vector that facilitates efficient delivery and expression of mCherry, or C-terminal mCherry fusions, to target cells. mCherry is a mutant fluorescent protein derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed (1). The excitation and emission maxima of the native mCherry protein are 587 nm and 610 nm, respectively.

The multiple cloning site (MCS) in pRetroQ-mCherry-C1 is located just downstream of the mCherry coding sequence. Genes cloned into the MCS are expressed as C-terminal mCherry fusion proteins when they are in the same reading frame as mCherry and there are no intervening stop codons.

The RetroQ retroviral vector backbone incorporates several unique features. This vector contains a puromycin resistance cassette (Puror) driven by the PGK promoter (PPGK) for selection of positively-infected cells (2).The hybrid 5’ long terminal repeats (LTR) consists of the CMV type I enhancer and the murine sarcoma virus (MSV) promoter. This vector demonstrates high levels of transcription in HEK 293-based packaging cell lines due, in part, to the presence of adenoviral E1A (3–6) in these cells. The self-inactivating feature of the vector is provided by a deletion in the 3’ LTR enhancer region (U3). During reverse transcription of the retroviral RNA, the inactivated 3’ LTR is copied and replaces the 5’ LTR CMV enhancer sequences. This can reduce the phenomenon known as promoter interference (7) and allow more efficient expression.

Additionally, the viral genomic transcript contains the necessary viral RNA processing elements, including the LTRs, packaging signal (ψ+), and tRNA primer-binding site. pRetroQ-mCherry-C1 contains a bacterial origin of replication, an E. coli Ampr gene for propagation and selection in bacteria, and an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen.

pRetroQ-mCherry-C1 is designed to efficiently deliver and express C-terminal mCherry fusions into primary cells or cells that are difficult to transfect. C-terminal mCherry fusion proteins retain the fluorescent properties of the native protein, allowing the in vivo localization of the fusion protein. The gene of interest should be cloned into pRetroQ-mCherry-C1 so that it is in frame with the mCherry coding sequence. The inserted sequence does not require an initiation codon (ATG) or a stop codon (TAA, TAG, TGA); however, if you don't want to use the stop codons downstream of the MCS (see map), you can add a stop codon to the end of your gene of interest. The recombinant mCherry vector can be transduced or transfected into mammalian cells. If required, stable transformants can be selected using puromycin. pRetroQ-mCherry-C1 can also be used simply to express mCherry in a cell line of interest (e.g., as an infection marker).

Before pRetroQ-mCherry-C1 can be transduced into mammalian cells, it must be transfected into a packaging cell line (such as the RetroPack PT67 Cell line, AmphoPack-293, EcoPack2-293, Pantropic Expression System , or Retro-X Universal Packaging System. The packaging cell line supplies the viral structural genes (gag, pol, and env) necessary for particle formation and replication that pRetroQ-mCherry-C1 lacks, allowing RNA from the vector to be packaged into non-infectious, replication-incompetent retroviral particles. Once a high-titer supernatant is produced, these retroviral particles can infect target cells and transmit the gene of interest, but they cannot replicate within the target 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 chances of producing replication-competent virus caused by recombination events during cell proliferation.

Propagation in E. coli

 Suitable host strains: DH5α, Fusion Blue, and other general purpose strains.

 Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.

 E. coli replication origin: ColE1

 Copy number: low

Excitation and emission maxima of mCherry

 Excitation maximum = 587 nm

 Emission maximum = 610 nm

pRetroQ-mCherry-C1载体序列：

ORIGIN

    1 TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC

   61 CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC

  121 GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA

  181 TGCCAAGTAC GCCCCCTATT GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC

  241 AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT CTACGTATTA GTCATCGCTA

  301 TTACCATGGT GATGCGGTTT TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC

  361 GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC

  421 AACGGGACTT TCCAAAATGT CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC

  481 GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTCAATAAAA GAGCCCACAA CCCCTCACTC

  541 GGCGCGCCAG TCTTCCGATA GACTGCGTCG CCCGGGTACC CGTATTCCCA ATAAAGCCTC

  601 TTGCTGTTTG CATCCGAATC GTGGTCTCGC TGTTCCTTGG GAGGGTCTCC TCTGAGTGAT

  661 TGACTACCCA CGACGGGGGT CTTTCATTTG GGGGCTCGTC CGGGATTTGG AGACCCCTGC

  721 CCAGGGACCA CCGACCCACC ACCGGGAGGT AAGCTGGCCA GCAACTTATC TGTGTCTGTC

  781 CGATTGTCTA GTGTCTATGT TTGATGTTAT GCGCCTGCGT CTGTACTAGT TAGCTAACTA

  841 GCTCTGTATC TGGCGGACCC GTGGTGGAAC TGACGAGTTC TGAACACCCG GCCGCAACCC

  901 TGGGAGACGT CCCAGGGACT TTGGGGGCCG TTTTTGTGGC CCGACCTGAG GAAGGGAGTC

  961 GATGTGGAAT CCGACCCCGT CAGGATATGT GGTTCTGGTA GGAGACGAGA ACCTAAAACA

 1021 GTTCCCGCCT CCGTCTGAAT TTTTGCTTTC GGTTTGGAAC CGAAGCCGCG CGTCTTGTCT

 1081 GCTGCAGCGC TGCAGCATCG TTCTGTGTTG TCTCTGTCTG ACTGTGTTTC TGTATTTGTC

 1141 TGAAAATTAG GGCCAGACTG TTACCACTCC CTTAAGTTTG ACCTTAGGTC ACTGGAAAGA

 1201 TGTCGAGCGG ATCGCTCACA ACCAGTCGGT AGATGTCAAG AAGAGACGTT GGGTTACCTT

 1261 CTGCTCTGCA GAATGGCCAA CCTTTAACGT CGGATGGCCG CGAGACGGCA CCTTTAACCG

 1321 AGACCTCATC ACCCAGGTTA AGATCAAGGT CTTTTCACCT GGCCCGCATG GACACCCAGA

 1381 CCAGGTCCCC TACATCGTGA CCTGGGAAGC CTTGGCTTTT GACCCCCCTC CCTGGGTCAA

 1441 GCCCTTTGTA CACCCTAAGC CTCCGCCTCC TCTTCCTCCA TCCGCCCCGT CTCTCCCCCT

 1501 TGAACCTCCT CGTTCGACCC CGCCTCGATC CTCCCTTTAT CCAGCCCTCA CTCCTTCTCT

 1561 AGGCGCCGGA ATTGAAGATC ATAGTTATTA ATAGTAATCA ATTACGGGGT CATTAGTTCA

 1621 TAGCCCATAT ATGGAGTTCC GCGTTACATA ACTTACGGTA AATGGCCCGC CTGGCTGACC

 1681 GCCCAACGAC CCCCGCCCAT TGACGTCAAT AATGACGTAT GTTCCCATAG TAACGCCAAT

 1741 AGGGACTTTC CATTGACGTC AATGGGTGGA GTATTTACGG TAAACTGCCC ACTTGGCAGT

 1801 ACATCAAGTG TATCATATGC CAAGTACGCC CCCTATTGAC GTCAATGACG GTAAATGGCC

 1861 CGCCTGGCAT TATGCCCAGT ACATGACCTT ATGGGACTTT CCTACTTGGC AGTACATCTA

 1921 CGTATTAGTC ATCGCTATTA CCATGGTGAT GCGGTTTTGG CAGTACATCA ATGGGCGTGG

 1981 ATAGCGGTTT GACTCACGGG GATTTCCAAG TCTCCACCCC ATTGACGTCA ATGGGAGTTT

 2041 GTTTTGGCAC CAAAATCAAC GGGACTTTCC AAAATGTCGT AACAACTCCG CCCCATTGAC

 2101 GCAAATGGGC GGTAGGCGTG TACGGTGGGA GGTCTATATA AGCAGAGCTG GTTTAGTGAA

 2161 CCGTCAGATC CGCTAGCGCT ACCGGTCGCC ACCATGGTGA GCAAGGGCGA GGAGGATAAC

 2221 ATGGCCATCA TCAAGGAGTT CATGCGCTTC AAGGTGCACA TGGAGGGCTC CGTGAACGGC

 2281 CACGAGTTCG AGATCGAGGG CGAGGGCGAG GGCCGCCCCT ACGAGGGCAC CCAGACCGCC

 2341 AAGCTGAAGG TGACCAAGGG TGGCCCCCTG CCCTTCGCCT GGGACATCCT GTCCCCTCAG

 2401 TTCATGTACG GCTCCAAGGC CTACGTGAAG CACCCCGCCG ACATCCCCGA CTACTTGAAG

 2461 CTGTCCTTCC CCGAGGGCTT CAAGTGGGAG CGCGTGATGA ACTTCGAGGA CGGCGGCGTG

 2521 GTGACCGTGA CCCAGGACTC CTCCCTGCAG GACGGCGAGT TCATCTACAA GGTGAAGCTG

 2581 CGCGGCACCA ACTTCCCCTC CGACGGCCCC GTAATGCAGA AGAAGACCAT GGGCTGGGAG

 2641 GCCTCCTCCG AGCGGATGTA CCCCGAGGAC GGCGCCCTGA AGGGCGAGAT CAAGCAGAGG

 2701 CTGAAGCTGA AGGACGGCGG CCACTACGAC GCTGAGGTCA AGACCACCTA CAAGGCCAAG

 2761 AAGCCCGTGC AGCTGCCCGG CGCCTACAAC GTCAACATCA AGTTGGACAT CACCTCCCAC

 2821 AACGAGGACT ACACCATCGT GGAACAGTAC GAACGCGCCG AGGGCCGCCA CTCCACCGGC

 2881 GGCATGGACG AGCTGTACAA GTCCGGACTC AGATCTCGAG CTCAAGCTTC GAATTCTGCA

 2941 GTCGACGGTA CCGCGGGCCC GGGATCCACC GGATCTAGAT AACTGATCAT AATTCTACCG

 3001 GGTAGGGGAG GCGCTTTTCC CAAGGCAGTC TGGAGCATGC GCTTTAGCAG CCCCGCTGGG

 3061 CACTTGGCGC TACACAAGTG GCCTCTGGCC TCGCACACAT TCCACATCCA CCGGTAGGCG

 3121 CCAACCGGCT CCGTTCTTTG GTGGCCCCTT CGCGCCACCT TCTACTCCTC CCCTAGTCAG

 3181 GAAGTTCCCC CCCGCCCCGC AGCTCGCGTC GTGCAGGACG TGACAAATGG AAGTAGCACG

 3241 TCTCACTAGT CTCGTGCAGA TGGACAGCAC CGCTGAGCAA TGGAAGCGGG TAGGCCTTTG

 3301 GGGCAGCGGC CAATAGCAGC TTTGCTCCTT CGCTTTCTGG GCTCAGAGGC TGGGAAGGGG

 3361 TGGGTCCGGG GGCGGGCTCA GGGGCGGGCT CAGGGGCGGG GCGGGCGCCC GAAGGTCCTC

 3421 CGGAGGCCCG GCATTCTGCA CGCTTCAAAA GCGCACGTCT GCCGCGCTGT TCTCCTCTTC

 3481 CTCATCTCCG GGCCTTTCGA CCTGCAGCCC AAGCTTACCA TGACCGAGTA CAAGCCCACG

 3541 GTGCGCCTCG CCACCCGCGA CGACGTCCCC AGGGCCGTAC GCACCCTCGC CGCCGCGTTC

 3601 GCCGACTACC CCGCCACGCG CCACACCGTC GATCCGGACC GCCACATCGA GCGGGTCACC

 3661 GAGCTGCAAG AACTCTTCCT CACGCGCGTC GGGCTCGACA TCGGCAAGGT GTGGGTCGCG

 3721 GACGACGGCG CCGCGGTGGC GGTCTGGACC ACGCCGGAGA GCGTCGAAGC GGGGGCGGTG

 3781 TTCGCCGAGA TCGGCCCGCG CATGGCCGAG TTGAGCGGTT CCCGGCTGGC CGCGCAGCAA

 3841 CAGATGGAAG GCCTCCTGGC GCCGCACCGG CCCAAGGAGC CCGCGTGGTT CCTGGCCACC

 3901 GTCGGCGTCT CGCCCGACCA CCAGGGCAAG GGTCTGGGCA GCGCCGTCGT GCTCCCCGGA

 3961 GTGGAGGCGG CCGAGCGCGC CGGGGTGCCC GCCTTCCTGG AGACCTCCGC GCCCCGCAAC

 4021 CTCCCCTTCT ACGAGCGGCT CGGCTTCACC GTCACCGCCG ACGTCGAGGT GCCCGAAGGA

 4081 CCGCGCACCT GGTGCATGAC CCGCAAGCCC GGTGCCTGAC GCCCGCCCCA CGACCCGCAG

 4141 CGCCCGACCG AAAGGAGCGC ACGACCCCAT GCATCGGCGT CTCGATCGAG ATATCAGTGG

 4201 TCCAGGCTCT AGTTTTGACT CAACAATATC ACCAGCTGAA GCCTATAGAG TACGAGCCAT

 4261 AGATAAAATA AAAGATTTTA TTTAGTCTCC AGAAAAAGGG GGGAATGAAA GACCCCACCT

 4321 GTAGGTTTGG CAAGCTACTA GCTTAAGTAA CGCCATTTTG CAAGGCATGG AAAAATACAT

 4381 AACTGAGAAT AGAGAAGTTC AGATCAAGGT CAGGAACAGA TGGAACAGGG TCGATCGACC

 4441 CTAGAGAACC ATCAGATGTT TCCAGGGTGC CCCAAGGACC TGAAATGACC CTGTGCCTTA

 4501 TTTGAACTAA CCAATCAGTT CGCTTCTCGC TTCTGTTCGC GCGCTTCTGC TCCCCGAGCT

 4561 CAATAAAAGA GCCCACAACC CCTCACTCGG GGCGCCAGTC CTCCGATTGA CTGAGTCGCC

 4621 CGGGTACCCG TGTATCCAAT AAACCCTCTT GCAGTTGCAT CCGACTTGTG GTCTCGCTGT

 4681 TCCTTGGGAG GGTCTCCTCT GAGTGATTGA CTACCCGTCA GCGGGGGTCT TTCATTTGGG

 4741 GGCTCGTCCG GGATCGGGAG ACCCCTGCCC AGGGACCACC GACCCACCAC CGGGAGGTAA

 4801 GCTGGCTGCC TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG

 4861 GAGACGGTCA CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG

 4921 TCAGCGGGTG TTGGCGGGTG TCGGGGCGCA GCCATGACCC AGTCACGTAG CGATAGCGGA

 4981 GTGTAGATCC GGCTGTGGAA TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA

 5041 GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCAGGTG TGGAAAGTCC

 5101 CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCATA

 5161 GTCCCGCCCC TAACTCCGCC CATCCCGCCC CTAACTCCGC CCAGTTCCGC CCATTCTCCG

 5221 CCCCATGGCT GACTAATTTT TTTTATTTAT GCAGAGGCCG AGGCCGCCTC GGCCTCTGAG

 5281 CTATTCCAGA AGTAGTGAGG AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA AAAGCTTACT

 5341 GGCTTAACTA TGCGGCATCA GAGCAGATTG TACTGAGAGT GCACCATATG CGGTGTGAAA

 5401 TACCGCACAG ATGCGTAAGG AGAAAATACC GCATCAGGCG CTCTTCCGCT TCCTCGCTCA

 5461 CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG

 5521 TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC

 5581 AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC

 5641 CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC

 5701 TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC

 5761 TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA

 5821 GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC

 5881 ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA

 5941 ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG

 6001 CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA

 6061 GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG

 6121 GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC

 6181 AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT

 6241 CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA

 6301 GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT

 6361 ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA

 6421 TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC

 6481 GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG

 6541 CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG

 6601 CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT

 6661 CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTGC AGGCATCGTG GTGTCACGCT

 6721 CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT

 6781 CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA

 6841 AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA

 6901 TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT

 6961 AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAC ACGGGATAAT ACCGCGCCAC

 7021 ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA

 7081 GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT

 7141 CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG

 7201 CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT

 7261 ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT

 7321 AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTCT

 7381 AAGAAACCAT TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG AGGCCCTTTC

 7441 GTCTTCAAGA ATTAGCTTGG CCATTGCATA CGTTGTATCC ATATCATAAT ATGTACATTT

 7501 ATATTGGCTC ATGTCCAACA TTACCGCCAT GTTGACATTG ATTATTGACT AGTTATTAAT

 7561 AGTAATCAAT TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGT

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