pRetroQ-mCherry-N1

pRetroQ-mCherry-N1

pRetroQ-mCherry-N1逆病毒载体基本信息：

pRetroQ-mCherry-N1载体质粒图谱和多克隆位点信息：

pRetroQ-mCherry-N1载体简介：

pRetroQ-mCherry-N1载体描述

pRetroQ-mCherry-N1 is a high-titer, self-inactivating retroviral vector that facilitates efficient delivery and expression of mCherry, or N-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 MCS in pRetroQ-mCherry-N1 lies between the human cytomegalovirus immediate-early promoter (PCMV IE) and the mCherry coding sequences. Genes cloned into the MCS are expressed as N-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 repeat (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 the5’ 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-N1 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-N1 is designed to efficiently deliver and express N-terminal mCherry fusions into primary cells or cells that are difficult to transfect. N-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-N1 so that it is in frame with the mCherry coding sequence. The inserted gene should include an initiation codon (ATG) and lack in-frame stop codons. The recombinant mCherry vector can be transduced or transfected into mammalian cells. If required, stable transformants can be selected using puromycin. pRetroQ-mCherry-N1 can also be used simply to express mCherry in a cell line of interest (e.g., as an infection marker).

Before pRetroQ-mCherry-N1 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-N1 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-N1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 ACCGGACTCA GATCTCGAGC TCAAGCTTCG AATTCTGCAG

 2221 TCGACGGTAC CGCGGGCCCG GGATCCACCG GTCATGGTGA GCAAGGGCGA GGAGGATAAC

 2281 ATGGCCATCA TCAAGGAGTT CATGCGCTTC AAGGTGCACA TGGAGGGCTC CGTGAACGGC

 2341 CACGAGTTCG AGATCGAGGG CGAGGGCGAG GGCCGCCCCT ACGAGGGCAC CCAGACCGCC

 2401 AAGCTGAAGG TGACCAAGGG TGGCCCCCTG CCCTTCGCCT GGGACATCCT GTCCCCTCAG

 2461 TTCATGTACG GCTCCAAGGC CTACGTGAAG CACCCCGCCG ACATCCCCGA CTACTTGAAG

 2521 CTGTCCTTCC CCGAGGGCTT CAAGTGGGAG CGCGTGATGA ACTTCGAGGA CGGCGGCGTG

 2581 GTGACCGTGA CCCAGGACTC CTCCCTGCAG GACGGCGAGT TCATCTACAA GGTGAAGCTG

 2641 CGCGGCACCA ACTTCCCCTC CGACGGCCCC GTAATGCAGA AGAAGACCAT GGGCTGGGAG

 2701 GCCTCCTCCG AGCGGATGTA CCCCGAGGAC GGCGCCCTGA AGGGCGAGAT CAAGCAGAGG

 2761 CTGAAGCTGA AGGACGGCGG CCACTACGAC GCTGAGGTCA AGACCACCTA CAAGGCCAAG

 2821 AAGCCCGTGC AGCTGCCCGG CGCCTACAAC GTCAACATCA AGTTGGACAT CACCTCCCAC

 2881 AACGAGGACT ACACCATCGT GGAACAGTAC GAACGCGCCG AGGGCCGCCA CTCCACCGGC

 2941 GGCATGGACG AGCTGTACAA GTGAGCGGCC GCGACTCTAG ATAATTCTAC CGGGTAGGGG

 3001 AGGCGCTTTT CCCAAGGCAG TCTGGAGCAT GCGCTTTAGC AGCCCCGCTG GGCACTTGGC

 3061 GCTACACAAG TGGCCTCTGG CCTCGCACAC ATTCCACATC CACCGGTAGG CGCCAACCGG

 3121 CTCCGTTCTT TGGTGGCCCC TTCGCGCCAC CTTCTACTCC TCCCCTAGTC AGGAAGTTCC

 3181 CCCCCGCCCC GCAGCTCGCG TCGTGCAGGA CGTGACAAAT GGAAGTAGCA CGTCTCACTA

 3241 GTCTCGTGCA GATGGACAGC ACCGCTGAGC AATGGAAGCG GGTAGGCCTT TGGGGCAGCG

 3301 GCCAATAGCA GCTTTGCTCC TTCGCTTTCT GGGCTCAGAG GCTGGGAAGG GGTGGGTCCG

 3361 GGGGCGGGCT CAGGGGCGGG CTCAGGGGCG GGGCGGGCGC CCGAAGGTCC TCCGGAGGCC

 3421 CGGCATTCTG CACGCTTCAA AAGCGCACGT CTGCCGCGCT GTTCTCCTCT TCCTCATCTC

 3481 CGGGCCTTTC GACCTGCAGC CCAAGCTTAC CATGACCGAG TACAAGCCCA CGGTGCGCCT

 3541 CGCCACCCGC GACGACGTCC CCAGGGCCGT ACGCACCCTC GCCGCCGCGT TCGCCGACTA

 3601 CCCCGCCACG CGCCACACCG TCGATCCGGA CCGCCACATC GAGCGGGTCA CCGAGCTGCA

 3661 AGAACTCTTC CTCACGCGCG TCGGGCTCGA CATCGGCAAG GTGTGGGTCG CGGACGACGG

 3721 CGCCGCGGTG GCGGTCTGGA CCACGCCGGA GAGCGTCGAA GCGGGGGCGG TGTTCGCCGA

 3781 GATCGGCCCG CGCATGGCCG AGTTGAGCGG TTCCCGGCTG GCCGCGCAGC AACAGATGGA

 3841 AGGCCTCCTG GCGCCGCACC GGCCCAAGGA GCCCGCGTGG TTCCTGGCCA CCGTCGGCGT

 3901 CTCGCCCGAC CACCAGGGCA AGGGTCTGGG CAGCGCCGTC GTGCTCCCCG GAGTGGAGGC

 3961 GGCCGAGCGC GCCGGGGTGC CCGCCTTCCT GGAGACCTCC GCGCCCCGCA ACCTCCCCTT

 4021 CTACGAGCGG CTCGGCTTCA CCGTCACCGC CGACGTCGAG GTGCCCGAAG GACCGCGCAC

 4081 CTGGTGCATG ACCCGCAAGC CCGGTGCCTG ACGCCCGCCC CACGACCCGC AGCGCCCGAC

 4141 CGAAAGGAGC GCACGACCCC ATGCATCGGC GTCTCGATCG AGATATCAGT GGTCCAGGCT

 4201 CTAGTTTTGA CTCAACAATA TCACCAGCTG AAGCCTATAG AGTACGAGCC ATAGATAAAA

 4261 TAAAAGATTT TATTTAGTCT CCAGAAAAAG GGGGGAATGA AAGACCCCAC CTGTAGGTTT

 4321 GGCAAGCTAC TAGCTTAAGT AACGCCATTT TGCAAGGCAT GGAAAAATAC ATAACTGAGA

 4381 ATAGAGAAGT TCAGATCAAG GTCAGGAACA GATGGAACAG GGTCGATCGA CCCTAGAGAA

 4441 CCATCAGATG TTTCCAGGGT GCCCCAAGGA CCTGAAATGA CCCTGTGCCT TATTTGAACT

 4501 AACCAATCAG TTCGCTTCTC GCTTCTGTTC GCGCGCTTCT GCTCCCCGAG CTCAATAAAA

 4561 GAGCCCACAA CCCCTCACTC GGGGCGCCAG TCCTCCGATT GACTGAGTCG CCCGGGTACC

 4621 CGTGTATCCA ATAAACCCTC TTGCAGTTGC ATCCGACTTG TGGTCTCGCT GTTCCTTGGG

 4681 AGGGTCTCCT CTGAGTGATT GACTACCCGT CAGCGGGGGT CTTTCATTTG GGGGCTCGTC

 4741 CGGGATCGGG AGACCCCTGC CCAGGGACCA CCGACCCACC ACCGGGAGGT AAGCTGGCTG

 4801 CCTCGCGCGT TTCGGTGATG ACGGTGAAAA CCTCTGACAC ATGCAGCTCC CGGAGACGGT

 4861 CACAGCTTGT CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CGTCAGGGCG CGTCAGCGGG

 4921 TGTTGGCGGG TGTCGGGGCG CAGCCATGAC CCAGTCACGT AGCGATAGCG GAGTGTAGAT

 4981 CCGGCTGTGG AATGTGTGTC AGTTAGGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG

 5041 AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCAGG TGTGGAAAGT CCCCAGGCTC

 5101 CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA TAGTCCCGCC

 5161 CCTAACTCCG CCCATCCCGC CCCTAACTCC GCCCAGTTCC GCCCATTCTC CGCCCCATGG

 5221 CTGACTAATT TTTTTTATTT ATGCAGAGGC CGAGGCCGCC TCGGCCTCTG AGCTATTCCA

 5281 GAAGTAGTGA GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC AAAAAGCTTA CTGGCTTAAC

 5341 TATGCGGCAT CAGAGCAGAT TGTACTGAGA GTGCACCATA TGCGGTGTGA AATACCGCAC

 5401 AGATGCGTAA GGAGAAAATA CCGCATCAGG CGCTCTTCCG CTTCCTCGCT CACTGACTCG

 5461 CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ACTCAAAGGC GGTAATACGG

 5521 TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACATGT GAGCAAAAGG CCAGCAAAAG

 5581 GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC

 5641 GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG ACTATAAAGA

 5701 TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT

 5761 ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA TAGCTCACGC

 5821 TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT GCACGAACCC

 5881 CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA

 5941 AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG AGCGAGGTAT

 6001 GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT ACGGCTACAC TAGAAGGACA

 6061 GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTTCG GAAAAAGAGT TGGTAGCTCT

 6121 TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA GCAGCAGATT

 6181 ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT

 6241 CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA GATTATCAAA AAGGATCTTC

 6301 ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAA TCTAAAGTAT ATATGAGTAA

 6361 ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGCAC CTATCTCAGC GATCTGTCTA

 6421 TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC

 6481 TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGACC CACGCTCACC GGCTCCAGAT

 6541 TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA

 6601 TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT

 6661 AATAGTTTGC GCAACGTTGT TGCCATTGCT GCAGGCATCG TGGTGTCACG CTCGTCGTTT

 6721 GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTACATG ATCCCCCATG

 6781 TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC

 6841 GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC

 6901 GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA ATAGTGTATG

 6961 CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ACACGGGATA ATACCGCGCC ACATAGCAGA

 7021 ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC AAGGATCTTA

 7081 CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT

 7141 TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG

 7201 GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCT TCCTTTTTCA ATATTATTGA

 7261 AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT

 7321 AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTGACGT CTAAGAAACC

 7381 ATTATTATCA TGACATTAAC CTATAAAAAT AGGCGTATCA CGAGGCCCTT TCGTCTTCAA

 7441 GAATTAGCTT GGCCATTGCA TACGTTGTAT CCATATCATA ATATGTACAT TTATATTGGC

 7501 TCATGTCCAA CATTACCGCC ATGTTGACAT TGATTATTGA CTAGTTATTA ATAGTAATCA

 7561 ATTACGGGGT CATTAGTTCA TAGCCCATAT ATGGAGT

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