pRetroQ-AcGFP1-C1

pRetroQ-AcGFP1-C1

pRetroQ-AcGFP1-C1逆病毒载体基本信息：

pRetroQ-AcGFP1-C1载体质粒图谱和多克隆位点信息：

pRetroQ-AcGFP1-C1载体简介：

pRetroQ-AcGFP1-C1载体描述

pRetroQ-AcGFP1-C1 is a high-titer, self-inactivating retroviral vector that facilitates efficient delivery and expression of AcGFP1, as well as C-terminal fusions of AcGFP1, to target cells. AcGFP1 is the green fluorescent protein from Aequorea coerulescens (AcGFP1; Excitation maximum = 475 nm; emission maximum = 505 nm).

The AcGFP1 coding sequence contains silent base changes, which correspond to human codon-usage preferences (1). The multiple cloning site (MCS) in pRetroQAcGFP1-C1 follows the AcGFP1 coding sequence. Genes cloned into the MCS are expressed as fusions to the C-terminus of AcGFP1 when they are in the same reading frame as AcGFP1 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-AcGFP1-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-AcGFP1-C1 is designed to efficiently deliver and express fusions to the C terminus of AcGFP1 into primary cells or cells that are difficult to transfect. Fusions to the C terminus of AcGFP1 retain the fluorescent properties of the native protein, allowing the in vivo localization of the fusion protein. The target gene should be cloned into pRetroQ- AcGFP1-C1 so that it is in frame with the AcGFP1 coding sequences, with no intervening in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant AcGFP1 vector can be infected or transfected into mammalian cells. If required, stable transformants can be selected using Puromycin. pRetroQ-AcGFP1-C1 can also be used simply to express AcGFP1 in a cell line of interest (e.g., as an infection marker).

Once pRetroQ-AcGFP1-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-AcGFP1-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-AcGFP1-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 GCAAGGGCGC CGAGCTGTTC

 2221 ACCGGCATCG TGCCCATCCT GATCGAGCTG AATGGCGATG TGAATGGCCA CAAGTTCAGC

 2281 GTGAGCGGCG AGGGCGAGGG CGATGCCACC TACGGCAAGC TGACCCTGAA GTTCATCTGC

 2341 ACCACCGGCA AGCTGCCTGT GCCCTGGCCC ACCCTGGTGA CCACCCTGAG CTACGGCGTG

 2401 CAGTGCTTCT CACGCTACCC CGATCACATG AAGCAGCACG ACTTCTTCAA GAGCGCCATG

 2461 CCTGAGGGCT ACATCCAGGA GCGCACCATC TTCTTCGAGG ATGACGGCAA CTACAAGTCG

 2521 CGCGCCGAGG TGAAGTTCGA GGGCGATACC CTGGTGAATC GCATCGAGCT GACCGGCACC

 2581 GATTTCAAGG AGGATGGCAA CATCCTGGGC AATAAGATGG AGTACAACTA CAACGCCCAC

 2641 AATGTGTACA TCATGACCGA CAAGGCCAAG AATGGCATCA AGGTGAACTT CAAGATCCGC

 2701 CACAACATCG AGGATGGCAG CGTGCAGCTG GCCGACCACT ACCAGCAGAA TACCCCCATC

 2761 GGCGATGGCC CTGTGCTGCT GCCCGATAAC CACTACCTGT CCACCCAGAG CGCCCTGTCC

 2821 AAGGACCCCA ACGAGAAGCG CGATCACATG ATCTACTTCG GCTTCGTGAC CGCCGCCGCC

 2881 ATCACCCACG GCATGGATGA GCTGTACAAG TCCGGACTCA GATCTCGAGC TCAAGCTTCG

 2941 AATTCTGCAG TCGACGGTAC CGCGGGCCCG GGATCCACCG GATCTAGATA ACTGATCATA

 3001 ATTCTACCGG GTAGGGGAGG CGCTTTTCCC AAGGCAGTCT GGAGCATGCG CTTTAGCAGC

 3061 CCCGCTGGGC ACTTGGCGCT ACACAAGTGG CCTCTGGCCT CGCACACATT CCACATCCAC

 3121 CGGTAGGCGC CAACCGGCTC CGTTCTTTGG TGGCCCCTTC GCGCCACCTT CTACTCCTCC

 3181 CCTAGTCAGG AAGTTCCCCC CCGCCCCGCA GCTCGCGTCG TGCAGGACGT GACAAATGGA

 3241 AGTAGCACGT CTCACTAGTC TCGTGCAGAT GGACAGCACC GCTGAGCAAT GGAAGCGGGT

 3301 AGGCCTTTGG GGCAGCGGCC AATAGCAGCT TTGCTCCTTC GCTTTCTGGG CTCAGAGGCT

 3361 GGGAAGGGGT GGGTCCGGGG GCGGGCTCAG GGGCGGGCTC AGGGGCGGGG CGGGCGCCCG

 3421 AAGGTCCTCC GGAGGCCCGG CATTCTGCAC GCTTCAAAAG CGCACGTCTG CCGCGCTGTT

 3481 CTCCTCTTCC TCATCTCCGG GCCTTTCGAC CTGCAGCCCA AGCTTACCAT GACCGAGTAC

 3541 AAGCCCACGG TGCGCCTCGC CACCCGCGAC GACGTCCCCA GGGCCGTACG CACCCTCGCC

 3601 GCCGCGTTCG CCGACTACCC CGCCACGCGC CACACCGTCG ATCCGGACCG CCACATCGAG

 3661 CGGGTCACCG AGCTGCAAGA ACTCTTCCTC ACGCGCGTCG GGCTCGACAT CGGCAAGGTG

 3721 TGGGTCGCGG ACGACGGCGC CGCGGTGGCG GTCTGGACCA CGCCGGAGAG CGTCGAAGCG

 3781 GGGGCGGTGT TCGCCGAGAT CGGCCCGCGC ATGGCCGAGT TGAGCGGTTC CCGGCTGGCC

 3841 GCGCAGCAAC AGATGGAAGG CCTCCTGGCG CCGCACCGGC CCAAGGAGCC CGCGTGGTTC

 3901 CTGGCCACCG TCGGCGTCTC GCCCGACCAC CAGGGCAAGG GTCTGGGCAG CGCCGTCGTG

 3961 CTCCCCGGAG TGGAGGCGGC CGAGCGCGCC GGGGTGCCCG CCTTCCTGGA GACCTCCGCG

 4021 CCCCGCAACC TCCCCTTCTA CGAGCGGCTC GGCTTCACCG TCACCGCCGA CGTCGAGGTG

 4081 CCCGAAGGAC CGCGCACCTG GTGCATGACC CGCAAGCCCG GTGCCTGACG CCCGCCCCAC

 4141 GACCCGCAGC GCCCGACCGA AAGGAGCGCA CGACCCCATG CATCGGCGTC TCGATCGAGA

 4201 TATCAGTGGT CCAGGCTCTA GTTTTGACTC AACAATATCA CCAGCTGAAG CCTATAGAGT

 4261 ACGAGCCATA GATAAAATAA AAGATTTTAT TTAGTCTCCA GAAAAAGGGG GGAATGAAAG

 4321 ACCCCACCTG TAGGTTTGGC AAGCTACTAG CTTAAGTAAC GCCATTTTGC AAGGCATGGA

 4381 AAAATACATA ACTGAGAATA GAGAAGTTCA GATCAAGGTC AGGAACAGAT GGAACAGGGT

 4441 CGATCGACCC TAGAGAACCA TCAGATGTTT CCAGGGTGCC CCAAGGACCT GAAATGACCC

 4501 TGTGCCTTAT TTGAACTAAC CAATCAGTTC GCTTCTCGCT TCTGTTCGCG CGCTTCTGCT

 4561 CCCCGAGCTC AATAAAAGAG CCCACAACCC CTCACTCGGG GCGCCAGTCC TCCGATTGAC

 4621 TGAGTCGCCC GGGTACCCGT GTATCCAATA AACCCTCTTG CAGTTGCATC CGACTTGTGG

 4681 TCTCGCTGTT CCTTGGGAGG GTCTCCTCTG AGTGATTGAC TACCCGTCAG CGGGGGTCTT

 4741 TCATTTGGGG GCTCGTCCGG GATCGGGAGA CCCCTGCCCA GGGACCACCG ACCCACCACC

 4801 GGGAGGTAAG CTGGCTGCCT CGCGCGTTTC GGTGATGACG GTGAAAACCT CTGACACATG

 4861 CAGCTCCCGG AGACGGTCAC AGCTTGTCTG TAAGCGGATG CCGGGAGCAG ACAAGCCCGT

 4921 CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT CGGGGCGCAG CCATGACCCA GTCACGTAGC

 4981 GATAGCGGAG TGTAGATCCG GCTGTGGAAT GTGTGTCAGT TAGGGTGTGG AAAGTCCCCA

 5041 GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCAGGTGT

 5101 GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA

 5161 GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC CAGTTCCGCC

 5221 CATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG CAGAGGCCGA GGCCGCCTCG

 5281 GCCTCTGAGC TATTCCAGAA GTAGTGAGGA GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA

 5341 AAGCTTACTG GCTTAACTAT GCGGCATCAG AGCAGATTGT ACTGAGAGTG CACCATATGC

 5401 GGTGTGAAAT ACCGCACAGA TGCGTAAGGA GAAAATACCG CATCAGGCGC TCTTCCGCTT

 5461 CCTCGCTCAC TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT

 5521 CAAAGGCGGT AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG

 5581 CAAAAGGCCA GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA

 5641 GGCTCCGCCC CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC

 5701 CGACAGGACT ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG

 5761 TTCCGACCCT GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC

 5821 TTTCTCATAG CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG

 5881 GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC

 5941 TTGAGTCCAA CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA

 6001 TTAGCAGAGC GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG

 6061 GCTACACTAG AAGGACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA

 6121 AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG

 6181 TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT

 6241 CTACGGGGTC TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT

 6301 TATCAAAAAG GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT

 6361 AAAGTATATA TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA

 6421 TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA

 6481 CTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCG CGAGACCCAC

 6541 GCTCACCGGC TCCAGATTTA TCAGCAATAA ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA

 6601 GTGGTCCTGC AACTTTATCC GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG

 6661 TAAGTAGTTC GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTGCA GGCATCGTGG

 6721 TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG

 6781 TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT CCGATCGTTG

 6841 TCAGAAGTAA GTTGGCCGCA GTGTTATCAC TCATGGTTAT GGCAGCACTG CATAATTCTC

 6901 TTACTGTCAT GCCATCCGTA AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCAT

 6961 TCTGAGAATA GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAACA CGGGATAATA

 7021 CCGCGCCACA TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA

 7081 AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA

 7141 ACTGATCTTC AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC

 7201 AAAATGCCGC AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC

 7261 TTTTTCAATA TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG

 7321 AATGTATTTA GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC

 7381 CTGACGTCTA AGAAACCATT ATTATCATGA CATTAACCTA TAAAAATAGG CGTATCACGA

 7441 GGCCCTTTCG TCTTCAAGAA TTAGCTTGGC CATTGCATAC GTTGTATCCA TATCATAATA

 7501 TGTACATTTA TATTGGCTCA TGTCCAACAT TACCGCCATG TTGACATTGA TTATTGACTA

 7561 GTTATTAATA GTAATCAATT ACGGGGTCAT TAGTTCATAG CCCATATATG GAGT//

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