pRetroQ-DsRed-Monomer-N1

pRetroQ-DsRed-Monomer-N1

pRetroQ-DsRed-Monomer-N1逆病毒载体基本信息：

pRetroQ-DsRed-Monomer-N1载体质粒图谱和多克隆位点信息：

pRetroQ-DsRed-Monomer-N1载体简介：

pRetroQ-DsRed Monomer-N1载体描述

pRetroQ-DsRed Monomer-N1 is a high-titer, self-inactivating retroviral vector that facilitates efficient delivery and expression of DsRed-monomer (DsRed.M1) as well as N 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-N1 lies between the immediate early promoter of CMV (PCMV IE) and the DsRed-Monomer coding sequences. Genes cloned into the MCS are expressed as fusions to the N-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. This vector contains a puromycin resistance cassette (Puror) driven by the PGK promoter 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 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. pRetroQDsRed Monomer-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 T antigen.

pRetroQ-DsRed Monomer-N1 is designed to efficiently deliver and express fusions to the N terminus of DsRed-Monomer into primary cells or cells that are difficult to transfect. Fusions to the N 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-N1 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-N1 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-N1 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-N1 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 replicationcompetent 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-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 GTCGCCACCA TGGACAACAC CGAGGACGTC

 2281 ATCAAGGAGT TCATGCAGTT CAAGGTGCGC ATGGAGGGCT CCGTGAACGG CCACTACTTC

 2341 GAGATCGAGG GCGAGGGCGA GGGCAAGCCC TACGAGGGCA CCCAGACCGC CAAGCTGCAG

 2401 GTGACCAAGG GCGGCCCCCT GCCCTTCGCC TGGGACATCC TGTCCCCCCA GTTCCAGTAC

 2461 GGCTCCAAGG CCTACGTGAA GCACCCCGCC GACATCCCCG ACTACATGAA GCTGTCCTTC

 2521 CCCGAGGGCT TCACCTGGGA GCGCTCCATG AACTTCGAGG ACGGCGGCGT GGTGGAGGTG

 2581 CAGCAGGACT CCTCCCTGCA GGACGGCACC TTCATCTACA AGGTGAAGTT CAAGGGCGTG

 2641 AACTTCCCCG CCGACGGCCC CGTAATGCAG AAGAAGACTG CCGGCTGGGA GCCCTCCACC

 2701 GAGAAGCTGT ACCCCCAGGA CGGCGTGCTG AAGGGCGAGA TCTCCCACGC CCTGAAGCTG

 2761 AAGGACGGCG GCCACTACAC CTGCGACTTC AAGACCGTGT ACAAGGCCAA GAAGCCCGTG

 2821 CAGCTGCCCG GCAACCACTA CGTGGACTCC AAGCTGGACA TCACCAACCA CAACGAGGAC

 2881 TACACCGTGG TGGAGCAGTA CGAGCACGCC GAGGCCCGCC ACTCCGGCTC CCAGTAGAGC

 2941 GGCCGCGACT CTAGATAATT CTACCGGGTA GGGGAGGCGC TTTTCCCAAG GCAGTCTGGA

 3001 GCATGCGCTT TAGCAGCCCC GCTGGGCACT TGGCGCTACA CAAGTGGCCT CTGGCCTCGC

 3061 ACACATTCCA CATCCACCGG TAGGCGCCAA CCGGCTCCGT TCTTTGGTGG CCCCTTCGCG

 3121 CCACCTTCTA CTCCTCCCCT AGTCAGGAAG TTCCCCCCCG CCCCGCAGCT CGCGTCGTGC

 3181 AGGACGTGAC AAATGGAAGT AGCACGTCTC ACTAGTCTCG TGCAGATGGA CAGCACCGCT

 3241 GAGCAATGGA AGCGGGTAGG CCTTTGGGGC AGCGGCCAAT AGCAGCTTTG CTCCTTCGCT

 3301 TTCTGGGCTC AGAGGCTGGG AAGGGGTGGG TCCGGGGGCG GGCTCAGGGG CGGGCTCAGG

 3361 GGCGGGGCGG GCGCCCGAAG GTCCTCCGGA GGCCCGGCAT TCTGCACGCT TCAAAAGCGC

 3421 ACGTCTGCCG CGCTGTTCTC CTCTTCCTCA TCTCCGGGCC TTTCGACCTG CAGCCCAAGC

 3481 TTACCATGAC CGAGTACAAG CCCACGGTGC GCCTCGCCAC CCGCGACGAC GTCCCCAGGG

 3541 CCGTACGCAC CCTCGCCGCC GCGTTCGCCG ACTACCCCGC CACGCGCCAC ACCGTCGATC

 3601 CGGACCGCCA CATCGAGCGG GTCACCGAGC TGCAAGAACT CTTCCTCACG CGCGTCGGGC

 3661 TCGACATCGG CAAGGTGTGG GTCGCGGACG ACGGCGCCGC GGTGGCGGTC TGGACCACGC

 3721 CGGAGAGCGT CGAAGCGGGG GCGGTGTTCG CCGAGATCGG CCCGCGCATG GCCGAGTTGA

 3781 GCGGTTCCCG GCTGGCCGCG CAGCAACAGA TGGAAGGCCT CCTGGCGCCG CACCGGCCCA

 3841 AGGAGCCCGC GTGGTTCCTG GCCACCGTCG GCGTCTCGCC CGACCACCAG GGCAAGGGTC

 3901 TGGGCAGCGC CGTCGTGCTC CCCGGAGTGG AGGCGGCCGA GCGCGCCGGG GTGCCCGCCT

 3961 TCCTGGAGAC CTCCGCGCCC CGCAACCTCC CCTTCTACGA GCGGCTCGGC TTCACCGTCA

 4021 CCGCCGACGT CGAGGTGCCC GAAGGACCGC GCACCTGGTG CATGACCCGC AAGCCCGGTG

 4081 CCTGACGCCC GCCCCACGAC CCGCAGCGCC CGACCGAAAG GAGCGCACGA CCCCATGCAT

 4141 CGGCGTCTCG ATCGAGATAT CAGTGGTCCA GGCTCTAGTT TTGACTCAAC AATATCACCA

 4201 GCTGAAGCCT ATAGAGTACG AGCCATAGAT AAAATAAAAG ATTTTATTTA GTCTCCAGAA

 4261 AAAGGGGGGA ATGAAAGACC CCACCTGTAG GTTTGGCAAG CTACTAGCTT AAGTAACGCC

 4321 ATTTTGCAAG GCATGGAAAA ATACATAACT GAGAATAGAG AAGTTCAGAT CAAGGTCAGG

 4381 AACAGATGGA ACAGGGTCGA TCGACCCTAG AGAACCATCA GATGTTTCCA GGGTGCCCCA

 4441 AGGACCTGAA ATGACCCTGT GCCTTATTTG AACTAACCAA TCAGTTCGCT TCTCGCTTCT

 4501 GTTCGCGCGC TTCTGCTCCC CGAGCTCAAT AAAAGAGCCC ACAACCCCTC ACTCGGGGCG

 4561 CCAGTCCTCC GATTGACTGA GTCGCCCGGG TACCCGTGTA TCCAATAAAC CCTCTTGCAG

 4621 TTGCATCCGA CTTGTGGTCT CGCTGTTCCT TGGGAGGGTC TCCTCTGAGT GATTGACTAC

 4681 CCGTCAGCGG GGGTCTTTCA TTTGGGGGCT CGTCCGGGAT CGGGAGACCC CTGCCCAGGG

 4741 ACCACCGACC CACCACCGGG AGGTAAGCTG GCTGCCTCGC GCGTTTCGGT GATGACGGTG

 4801 AAAACCTCTG ACACATGCAG CTCCCGGAGA CGGTCACAGC TTGTCTGTAA GCGGATGCCG

 4861 GGAGCAGACA AGCCCGTCAG GGCGCGTCAG CGGGTGTTGG CGGGTGTCGG GGCGCAGCCA

 4921 TGACCCAGTC ACGTAGCGAT AGCGGAGTGT AGATCCGGCT GTGGAATGTG TGTCAGTTAG

 4981 GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT

 5041 AGTCAGCAAC CAGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA

 5101 TGCATCTCAA TTAGTCAGCA ACCATAGTCC CGCCCCTAAC TCCGCCCATC CCGCCCCTAA

 5161 CTCCGCCCAG TTCCGCCCAT TCTCCGCCCC ATGGCTGACT AATTTTTTTT ATTTATGCAG

 5221 AGGCCGAGGC CGCCTCGGCC TCTGAGCTAT TCCAGAAGTA GTGAGGAGGC TTTTTTGGAG

 5281 GCCTAGGCTT TTGCAAAAAG CTTACTGGCT TAACTATGCG GCATCAGAGC AGATTGTACT

 5341 GAGAGTGCAC CATATGCGGT GTGAAATACC GCACAGATGC GTAAGGAGAA AATACCGCAT

 5401 CAGGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG

 5461 AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC

 5521 AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT

 5581 GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG

 5641 TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC

 5701 CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC

 5761 TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT

 5821 CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT

 5881 ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC

 5941 AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA

 6001 GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG CTCTGCTGAA

 6061 GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG

 6121 TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA

 6181 AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG

 6241 GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG

 6301 AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT

 6361 AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT

 6421 CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT

 6481 GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG

 6541 AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG

 6601 TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT

 6661 TGCTGCAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC

 6721 CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT

 6781 CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC

 6841 AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA

 6901 GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC

 6961 GTCAACACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA

 7021 ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA

 7081 ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG

 7141 AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG

 7201 AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT

 7261 GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT

 7321 TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT TAACCTATAA

 7381 AAATAGGCGT ATCACGAGGC CCTTTCGTCT TCAAGAATTA GCTTGGCCAT TGCATACGTT

 7441 GTATCCATAT CATAATATGT ACATTTATAT TGGCTCATGT CCAACATTAC CGCCATGTTG

 7501 ACATTGATTA TTGACTAGTT ATTAATAGTA ATCAATTACG GGGTCATTAG TTCATAGCCC

 7561 ATATATGGAG T

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