pRetroQ-DsRed-Monomer-C1

pRetroQ-DsRed-Monomer-C1

pRetroQ-DsRed-Monomer-C1逆病毒载体基本信息：

pRetroQ-DsRed-Monomer-C1载体质粒图谱和多克隆位点信息：

pRetroQ-DsRed-Monomer-C1载体简介：

pRetroQ-DsRed-Monomer-C1载体描述

pRetroQ-DsRed-Monomer-C1 is a high-titer, self-inactivating retroviral vector that facilitates efficient delivery and expression of DsRed-monomer (DsRed.M1) as well as C terminal fusions of DsRed monomer to target cells. DsRed.M1 is a monomeric mutant derived from the tetrameric Discosoma sp. red fluorescent protein DsRed (1).

DsRed-Monomer contains forty-five amino acid substitutions. When DsRed-Monomer is expressed in mammalian cell cultures, red fluorescent cells can be detected by either fluorescence microscopy or flow cytometry 12–16 hours after transfection or 24–48 hours after infection (DsRed-Monomer excitation and emission maxima = 557 nm and 592 nm, respectively). The DsRed-Monomer coding sequence is human codon-optimized for high expression in mammalian cells (2) The MCS in pRetroQ-DsRed Monomer-C1 follows the DsRed-Monomer coding sequences. Genes cloned into the MCS are expressed as fusions to the C-terminus of DsRed Monomer when they are in the same reading frame as DsRed Monomer and there are no intervening stop codons.

The RetroQ retroviral vector backbone incorporates several unique features. 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 the 5’ LTR CMV enhancer sequences. This can reduce the phenomenon known as promoter interference (7) and allow more efficient expression. This vector also contains a puromycin resistance cassette (Puror) driven by the PGK promoter for selection of positively infected cells (2).

Additionally, the viral genomic transcript contains the necessary viral RNA processing elements including the LTRs, packaging signal (ψ+), and tRNA primer-binding site. pRetroQ-DsRed Monomer-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 T antigen.

pRetro-DsRed Monomer-C1 is designed to efficiently deliver and express fusions to the C terminus of DsRed-Monomer into primary cells or cells that are difficult to transfect. Fusions to the C terminus of DsRed-Monomer retain the fluorescent properties of the native protein, allowing the in vivo localization of the fusion protein. The target gene should be cloned into pDsRed Monomer-C1 so that it is in frame with the DsRed-Monomer coding sequences, with no intervening in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant DsRed Monomer vector can be infected or transfected into mammalian cells.

If required, stable transformants can be selected using Puromycin. pRetroQ DsRed Monomer-C1 can also be used simply to express DsRed Monomer in a cell line of interest (e.g., as an infection marker).Once pRetroQ-DsRed Monomer-C1 is 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, RNA from the vector is packaged into non-infectious, replication-incompetent retroviral particles, since pRetroQ-DsRed Monomer-C1 lacks the structural genes (gag, pol, and env) necessary for particle formation and replication. These genes, however, are stably integrated as part of the packaging cell genome. Once a high-titer supernatant is produced, these 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 chances of producing replication-competent virus due to 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

pRetroQ-DsRed-Monomer-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 GCCCGCTCTG GCATTATGCC

  241 CAGTACATGA CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTACGTATT AGTCATCGCT

  301 ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG GTTTGACTCA

  361 CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA GTTTGTTTTG GCACCAAAAT

  421 CAACGGGACT TTCCAAAATG TCGTAACAAC TCCGCCCCAT TGACGCAAAT GGGCGGTAGG

  481 CGTGTACGGT GGGAGGTCTA TATAAGCAGA GCTCAATAAA AGAGCCCACA ACCCCTCACT

  541 CGGCGCGCCA GTCTTCCGAT AGACTGCGTC GCCCGGGTAC CCGTATTCCC AATAAAGCCT

  601 CTTGCTGTTT GCATCCGAAT CGTGGTCTCG CTGTTCCTTG GGAGGGTCTC CTCTGAGTGA

  661 TTGACTACCC ACGACGGGGG TCTTTCATTT GGGGGCTCGT CCGGGATTTG GAGACCCCTG

  721 CCCAGGGACC ACCGACCCAC CACCGGGAGG TAAGCTGGCC AGCAACTTAT CTGTGTCTGT

  781 CCGATTGTCT AGTGTCTATG TTTGATGTTA TGCGCCTGCG TCTGTACTAG TTAGCTAACT

  841 AGCTCTGTAT CTGGCGGACC CGTGGTGGAA CTGACGAGTT CTGAACACCC GGCCGCAACC

  901 CTGGGAGACG TCCCAGGGAC TTTGGGGGCC GTTTTTGTGG CCCGACCTGA GGAAGGGAGT

  961 CGATGTGGAA TCCGACCCCG TCAGGATATG TGGTTCTGGT AGGAGACGAG AACCTAAAAC

 1021 AGTTCCCGCC TCCGTCTGAA TTTTTGCTTT CGGTTTGGAA CCGAAGCCGC GCGTCTTGTC

 1081 TGCTGCAGCG CTGCAGCATC GTTCTGTGTT GTCTCTGTCT GACTGTGTTT CTGTATTTGT

 1141 CTGAAAATTA GGGCCAGACT GTTACCACTC CCTTAAGTTT GACCTTAGGT CACTGGAAAG

 1201 ATGTCGAGCG GATCGCTCAC AACCAGTCGG TAGATGTCAA GAAGAGACGT TGGGTTACCT

 1261 TCTGCTCTGC AGAATGGCCA ACCTTTAACG TCGGATGGCC GCGAGACGGC ACCTTTAACC

 1321 GAGACCTCAT CACCCAGGTT AAGATCAAGG TCTTTTCACC TGGCCCGCAT GGACACCCAG

 1381 ACCAGGTCCC CTACATCGTG ACCTGGGAAG CCTTGGCTTT TGACCCCCCT CCCTGGGTCA

 1441 AGCCCTTTGT ACACCCTAAG CCTCCGCCTC CTCTTCCTCC ATCCGCCCCG TCTCTCCCCC

 1501 TTGAACCTCC TCGTTCGACC CCGCCTCGAT CCTCCCTTTA TCCAGCCCTC ACTCCTTCTC

 1561 TAGGCGCCGG AATTGAAGAT CATAGTTATT AATAGTAATC AATTACGGGG TCATTAGTTC

 1621 ATAGCCCATA TATGGAGTTC CGCGTTACAT AACTTACGGT AAATGGCCCG CCTGGCTGAC

 1681 CGCCCAACGA CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA

 1741 TAGGGACTTT CCATTGACGT CAATGGGTGG AGTATTTACG GTAAACTGCC CACTTGGCAG

 1801 TACATCAAGT GTATCATATG CCAAGTACGC CCCCTATTGA CGTCAATGAC GGTAAATGGC

 1861 CCGCCTGGCA TTATGCCCAG TACATGACCT TATGGGACTT TCCTACTTGG CAGTACATCT

 1921 ACGTATTAGT CATCGCTATT ACCATGGTGA TGCGGTTTTG GCAGTACATC AATGGGCGTG

 1981 GATAGCGGTT TGACTCACGG GGATTTCCAA GTCTCCACCC CATTGACGTC AATGGGAGTT

 2041 TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG TAACAACTCC GCCCCATTGA

 2101 CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT AAGCAGAGCT GGTTTAGTGA

 2161 ACCGTCAGAT CCGCTAGCGC TACCGGTCGC CACCATGGAC AACACCGAGG ACGTCATCAA

 2221 GGAGTTCATG CAGTTCAAGG TGCGCATGGA GGGCTCCGTG AACGGCCACT ACTTCGAGAT

 2281 CGAGGGCGAG GGCGAGGGCA AGCCCTACGA GGGCACCCAG ACCGCCAAGC TGCAGGTGAC

 2341 CAAGGGCGGC CCCCTGCCCT TCGCCTGGGA CATCCTGTCC CCCCAGTTCC AGTACGGCTC

 2401 CAAGGCCTAC GTGAAGCACC CCGCCGACAT CCCCGACTAC ATGAAGCTGT CCTTCCCCGA

 2461 GGGCTTCACC TGGGAGCGCT CCATGAACTT CGAGGACGGC GGCGTGGTGG AGGTGCAGCA

 2521 GGACTCCTCC CTGCAGGACG GCACCTTCAT CTACAAGGTG AAGTTCAAGG GCGTGAACTT

 2581 CCCCGCCGAC GGCCCCGTAA TGCAGAAGAA GACTGCCGGC TGGGAGCCCT CCACCGAGAA

 2641 GCTGTACCCC CAGGACGGCG TGCTGAAGGG CGAGATCTCC CACGCCCTGA AGCTGAAGGA

 2701 CGGCGGCCAC TACACCTGCG ACTTCAAGAC CGTGTACAAG GCCAAGAAGC CCGTGCAGCT

 2761 GCCCGGCAAC CACTACGTGG ACTCCAAGCT GGACATCACC AACCACAACG AGGACTACAC

 2821 CGTGGTGGAG CAGTACGAGC ACGCCGAGGC CCGCCACTCC GGCTCCCAGT CCGGACTCAG

 2881 ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGG GATCCACCGG

 2941 ATCTAGATAA CTGATCATAA TTCTACCGGG TAGGGGAGGC GCTTTTCCCA AGGCAGTCTG

 3001 GAGCATGCGC TTTAGCAGCC CCGCTGGGCA CTTGGCGCTA CACAAGTGGC CTCTGGCCTC

 3061 GCACACATTC CACATCCACC GGTAGGCGCC AACCGGCTCC GTTCTTTGGT GGCCCCTTCG

 3121 CGCCACCTTC TACTCCTCCC CTAGTCAGGA AGTTCCCCCC CGCCCCGCAG CTCGCGTCGT

 3181 GCAGGACGTG ACAAATGGAA GTAGCACGTC TCACTAGTCT CGTGCAGATG GACAGCACCG

 3241 CTGAGCAATG GAAGCGGGTA GGCCTTTGGG GCAGCGGCCA ATAGCAGCTT TGCTCCTTCG

 3301 CTTTCTGGGC TCAGAGGCTG GGAAGGGGTG GGTCCGGGGG CGGGCTCAGG GGCGGGCTCA

 3361 GGGGCGGGGC GGGCGCCCGA AGGTCCTCCG GAGGCCCGGC ATTCTGCACG CTTCAAAAGC

 3421 GCACGTCTGC CGCGCTGTTC TCCTCTTCCT CATCTCCGGG CCTTTCGACC TGCAGCCCAA

 3481 GCTTACCATG ACCGAGTACA AGCCCACGGT GCGCCTCGCC ACCCGCGACG ACGTCCCCAG

 3541 GGCCGTACGC ACCCTCGCCG CCGCGTTCGC CGACTACCCC GCCACGCGCC ACACCGTCGA

 3601 TCCGGACCGC CACATCGAGC GGGTCACCGA GCTGCAAGAA CTCTTCCTCA CGCGCGTCGG

 3661 GCTCGACATC GGCAAGGTGT GGGTCGCGGA CGACGGCGCC GCGGTGGCGG TCTGGACCAC

 3721 GCCGGAGAGC GTCGAAGCGG GGGCGGTGTT CGCCGAGATC GGCCCGCGCA TGGCCGAGTT

 3781 GAGCGGTTCC CGGCTGGCCG CGCAGCAACA GATGGAAGGC CTCCTGGCGC CGCACCGGCC

 3841 CAAGGAGCCC GCGTGGTTCC TGGCCACCGT CGGCGTCTCG CCCGACCACC AGGGCAAGGG

 3901 TCTGGGCAGC GCCGTCGTGC TCCCCGGAGT GGAGGCGGCC GAGCGCGCCG GGGTGCCCGC

 3961 CTTCCTGGAG ACCTCCGCGC CCCGCAACCT CCCCTTCTAC GAGCGGCTCG GCTTCACCGT

 4021 CACCGCCGAC GTCGAGGTGC CCGAAGGACC GCGCACCTGG TGCATGACCC GCAAGCCCGG

 4081 TGCCTGACGC CCGCCCCACG ACCCGCAGCG CCCGACCGAA AGGAGCGCAC GACCCCATGC

 4141 ATCGGCGTCT CGATCGAGAT ATCAGTGGTC CAGGCTCTAG TTTTGACTCA ACAATATCAC

 4201 CAGCTGAAGC CTATAGAGTA CGAGCCATAG ATAAAATAAA AGATTTTATT TAGTCTCCAG

 4261 AAAAAGGGGG GAATGAAAGA CCCCACCTGT AGGTTTGGCA AGCTACTAGC TTAAGTAACG

 4321 CCATTTTGCA AGGCATGGAA AAATACATAA CTGAGAATAG AGAAGTTCAG ATCAAGGTCA

 4381 GGAACAGATG GAACAGGGTC GATCGACCCT AGAGAACCAT CAGATGTTTC CAGGGTGCCC

 4441 CAAGGACCTG AAATGACCCT GTGCCTTATT TGAACTAACC AATCAGTTCG CTTCTCGCTT

 4501 CTGTTCGCGC GCTTCTGCTC CCCGAGCTCA ATAAAAGAGC CCACAACCCC TCACTCGGGG

 4561 CGCCAGTCCT CCGATTGACT GAGTCGCCCG GGTACCCGTG TATCCAATAA ACCCTCTTGC

 4621 AGTTGCATCC GACTTGTGGT CTCGCTGTTC CTTGGGAGGG TCTCCTCTGA GTGATTGACT

 4681 ACCCGTCAGC GGGGGTCTTT CATTTGGGGG CTCGTCCGGG ATCGGGAGAC CCCTGCCCAG

 4741 GGACCACCGA CCCACCACCG GGAGGTAAGC TGGCTGCCTC GCGCGTTTCG GTGATGACGG

 4801 TGAAAACCTC TGACACATGC AGCTCCCGGA GACGGTCACA GCTTGTCTGT AAGCGGATGC

 4861 CGGGAGCAGA CAAGCCCGTC AGGGCGCGTC AGCGGGTGTT GGCGGGTGTC GGGGCGCAGC

 4921 CATGACCCAG TCACGTAGCG ATAGCGGAGT GTAGATCCGG CTGTGGAATG TGTGTCAGTT

 4981 AGGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA TGCATCTCAA

 5041 TTAGTCAGCA ACCAGGTGTG GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA GTATGCAAAG

 5101 CATGCATCTC AATTAGTCAG CAACCATAGT CCCGCCCCTA ACTCCGCCCA TCCCGCCCCT

 5161 AACTCCGCCC AGTTCCGCCC ATTCTCCGCC CCATGGCTGA CTAATTTTTT TTATTTATGC

 5221 AGAGGCCGAG GCCGCCTCGG CCTCTGAGCT ATTCCAGAAG TAGTGAGGAG GCTTTTTTGG

 5281 AGGCCTAGGC TTTTGCAAAA AGCTTACTGG CTTAACTATG CGGCATCAGA GCAGATTGTA

 5341 CTGAGAGTGC ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC

 5401 ATCAGGCGCT CTTCCGCTTC CTCGCTCACT GACTCGCTGC GCTCGGTCGT TCGGCTGCGG

 5461 CGAGCGGTAT CAGCTCACTC AAAGGCGGTA ATACGGTTAT CCACAGAATC AGGGGATAAC

 5521 GCAGGAAAGA ACATGTGAGC AAAAGGCCAG CAAAAGGCCA GGAACCGTAA AAAGGCCGCG

 5581 TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC ATCACAAAAA TCGACGCTCA

 5641 AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC AGGCGTTTCC CCCTGGAAGC

 5701 TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC CGCCTTTCTC

 5761 CCTTCGGGAA GCGTGGCGCT TTCTCATAGC TCACGCTGTA GGTATCTCAG TTCGGTGTAG

 5821 GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG TTCAGCCCGA CCGCTGCGCC

 5881 TTATCCGGTA ACTATCGTCT TGAGTCCAAC CCGGTAAGAC ACGACTTATC GCCACTGGCA

 5941 GCAGCCACTG GTAACAGGAT TAGCAGAGCG AGGTATGTAG GCGGTGCTAC AGAGTTCTTG

 6001 AAGTGGTGGC CTAACTACGG CTACACTAGA AGGACAGTAT TTGGTATCTG CGCTCTGCTG

 6061 AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT CCGGCAAACA AACCACCGCT

 6121 GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG CAGATTACGC GCAGAAAAAA AGGATCTCAA

 6181 GAAGATCCTT TGATCTTTTC TACGGGGTCT GACGCTCAGT GGAACGAAAA CTCACGTTAA

 6241 GGGATTTTGG TCATGAGATT ATCAAAAAGG ATCTTCACCT AGATCCTTTT AAATTAAAAA

 6301 TGAAGTTTTA AATCAATCTA AAGTATATAT GAGTAAACTT GGTCTGACAG TTACCAATGC

 6361 TTAATCAGTG AGGCACCTAT CTCAGCGATC TGTCTATTTC GTTCATCCAT AGTTGCCTGA

 6421 CTCCCCGTCG TGTAGATAAC TACGATACGG GAGGGCTTAC CATCTGGCCC CAGTGCTGCA

 6481 ATGATACCGC GAGACCCACG CTCACCGGCT CCAGATTTAT CAGCAATAAA CCAGCCAGCC

 6541 GGAAGGGCCG AGCGCAGAAG TGGTCCTGCA ACTTTATCCG CCTCCATCCA GTCTATTAAT

 6601 TGTTGCCGGG AAGCTAGAGT AAGTAGTTCG CCAGTTAATA GTTTGCGCAA CGTTGTTGCC

 6661 ATTGCTGCAG GCATCGTGGT GTCACGCTCG TCGTTTGGTA TGGCTTCATT CAGCTCCGGT

 6721 TCCCAACGAT CAAGGCGAGT TACATGATCC CCCATGTTGT GCAAAAAAGC GGTTAGCTCC

 6781 TTCGGTCCTC CGATCGTTGT CAGAAGTAAG TTGGCCGCAG TGTTATCACT CATGGTTATG

 6841 GCAGCACTGC ATAATTCTCT TACTGTCATG CCATCCGTAA GATGCTTTTC TGTGACTGGT

 6901 GAGTACTCAA CCAAGTCATT CTGAGAATAG TGTATGCGGC GACCGAGTTG CTCTTGCCCG

 6961 GCGTCAACAC GGGATAATAC CGCGCCACAT AGCAGAACTT TAAAAGTGCT CATCATTGGA

 7021 AAACGTTCTT CGGGGCGAAA ACTCTCAAGG ATCTTACCGC TGTTGAGATC CAGTTCGATG

 7081 TAACCCACTC GTGCACCCAA CTGATCTTCA GCATCTTTTA CTTTCACCAG CGTTTCTGGG

 7141 TGAGCAAAAA CAGGAAGGCA AAATGCCGCA AAAAAGGGAA TAAGGGCGAC ACGGAAATGT

 7201 TGAATACTCA TACTCTTCCT TTTTCAATAT TATTGAAGCA TTTATCAGGG TTATTGTCTC

 7261 ATGAGCGGAT ACATATTTGA ATGTATTTAG AAAAATAAAC AAATAGGGGT TCCGCGCACA

 7321 TTTCCCCGAA AAGTGCCACC TGACGTCTAA GAAACCATTA TTATCATGAC ATTAACCTAT

 7381 AAAAATAGGC GTATCACGAG GCCCTTTCGT CTTCAAGAAT TAGCTTGGCC ATTGCATACG

 7441 TTGTATCCAT ATCATAATAT GTACATTTAT ATTGGCTCAT GTCCAACATT ACCGCCATGT

 7501 TGACATTGAT TATTGACTAG TTATTAATAG TAATCAATTA CGGGGTCATT AGTTCATAGC

 7561 CCATATATGG AGT

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