pRetroQ-AcGFP1-N1

pRetroQ-AcGFP1-N1

pRetroQ-AcGFP1-N1逆病毒载体基本信息：

pRetroQ-AcGFP1-N1载体质粒图谱和多克隆位点信息：

pRetroQ-AcGFP1-N1载体简介：

pRetroQ-AcGFP1-N1载体描述

pRetroQ-AcGFP1-N1 is a high-titer, self-inactivating retroviral vector that facilitates efficient delivery and expression of AcGFP1, as well as N 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 coding sequence of the AcGFP1 gene contains silent base changes, which correspond to human codon-usage preferences (1). The MCS in pRetroQAcGFP1-N1 lies between the immediate early promoter of CMV (PCMV IE) and the AcGFP1 coding sequences. Genes cloned into the MCS are expressed as fusions to the N-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 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 efficientexpression.

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-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-AcGFP1-N1 is designed to efficiently deliver and express fusions to the N terminus of AcGFP1 into primary cells or cells that are difficult to transfect. Fusions to the N 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-N1 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-N1 can also be used simply to express AcGFP1 in a cell line of interest (e.g., as an infection marker).

Once pRetroQ-AcGFP1-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-AcGFP1-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 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-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 GCAAGGGCGC CGAGCTGTTC

 2281 ACCGGCATCG TGCCCATCCT GATCGAGCTG AATGGCGATG TGAATGGCCA CAAGTTCAGC

 2341 GTGAGCGGCG AGGGCGAGGG CGATGCCACC TACGGCAAGC TGACCCTGAA GTTCATCTGC

 2401 ACCACCGGCA AGCTGCCTGT GCCCTGGCCC ACCCTGGTGA CCACCCTGAG CTACGGCGTG

 2461 CAGTGCTTCT CACGCTACCC CGATCACATG AAGCAGCACG ACTTCTTCAA GAGCGCCATG

 2521 CCTGAGGGCT ACATCCAGGA GCGCACCATC TTCTTCGAGG ATGACGGCAA CTACAAGTCG

 2581 CGCGCCGAGG TGAAGTTCGA GGGCGATACC CTGGTGAATC GCATCGAGCT GACCGGCACC

 2641 GATTTCAAGG AGGATGGCAA CATCCTGGGC AATAAGATGG AGTACAACTA CAACGCCCAC

 2701 AATGTGTACA TCATGACCGA CAAGGCCAAG AATGGCATCA AGGTGAACTT CAAGATCCGC

 2761 CACAACATCG AGGATGGCAG CGTGCAGCTG GCCGACCACT ACCAGCAGAA TACCCCCATC

 2821 GGCGATGGCC CTGTGCTGCT GCCCGATAAC CACTACCTGT CCACCCAGAG CGCCCTGTCC

 2881 AAGGACCCCA ACGAGAAGCG CGATCACATG ATCTACTTCG GCTTCGTGAC CGCCGCCGCC

 2941 ATCACCCACG GCATGGATGA GCTGTACAAG TGAGCGGCCG CGACTCTAGA TAATTCTACC

 3001 GGGTAGGGGA GGCGCTTTTC CCAAGGCAGT CTGGAGCATG CGCTTTAGCA GCCCCGCTGG

 3061 GCACTTGGCG CTACACAAGT GGCCTCTGGC CTCGCACACA TTCCACATCC ACCGGTAGGC

 3121 GCCAACCGGC TCCGTTCTTT GGTGGCCCCT TCGCGCCACC TTCTACTCCT CCCCTAGTCA

 3181 GGAAGTTCCC CCCCGCCCCG CAGCTCGCGT CGTGCAGGAC GTGACAAATG GAAGTAGCAC

 3241 GTCTCACTAG TCTCGTGCAG ATGGACAGCA CCGCTGAGCA ATGGAAGCGG GTAGGCCTTT

 3301 GGGGCAGCGG CCAATAGCAG CTTTGCTCCT TCGCTTTCTG GGCTCAGAGG CTGGGAAGGG

 3361 GTGGGTCCGG GGGCGGGCTC AGGGGCGGGC TCAGGGGCGG GGCGGGCGCC CGAAGGTCCT

 3421 CCGGAGGCCC GGCATTCTGC ACGCTTCAAA AGCGCACGTC TGCCGCGCTG TTCTCCTCTT

 3481 CCTCATCTCC GGGCCTTTCG ACCTGCAGCC CAAGCTTACC ATGACCGAGT ACAAGCCCAC

 3541 GGTGCGCCTC GCCACCCGCG ACGACGTCCC CAGGGCCGTA CGCACCCTCG CCGCCGCGTT

 3601 CGCCGACTAC CCCGCCACGC GCCACACCGT CGATCCGGAC CGCCACATCG AGCGGGTCAC

 3661 CGAGCTGCAA GAACTCTTCC TCACGCGCGT CGGGCTCGAC ATCGGCAAGG TGTGGGTCGC

 3721 GGACGACGGC GCCGCGGTGG CGGTCTGGAC CACGCCGGAG AGCGTCGAAG CGGGGGCGGT

 3781 GTTCGCCGAG ATCGGCCCGC GCATGGCCGA GTTGAGCGGT TCCCGGCTGG CCGCGCAGCA

 3841 ACAGATGGAA GGCCTCCTGG CGCCGCACCG GCCCAAGGAG CCCGCGTGGT TCCTGGCCAC

 3901 CGTCGGCGTC TCGCCCGACC ACCAGGGCAA GGGTCTGGGC AGCGCCGTCG TGCTCCCCGG

 3961 AGTGGAGGCG GCCGAGCGCG CCGGGGTGCC CGCCTTCCTG GAGACCTCCG CGCCCCGCAA

 4021 CCTCCCCTTC TACGAGCGGC TCGGCTTCAC CGTCACCGCC GACGTCGAGG TGCCCGAAGG

 4081 ACCGCGCACC TGGTGCATGA CCCGCAAGCC CGGTGCCTGA CGCCCGCCCC ACGACCCGCA

 4141 GCGCCCGACC GAAAGGAGCG CACGACCCCA TGCATCGGCG TCTCGATCGA GATATCAGTG

 4201 GTCCAGGCTC TAGTTTTGAC TCAACAATAT CACCAGCTGA AGCCTATAGA GTACGAGCCA

 4261 TAGATAAAAT AAAAGATTTT ATTTAGTCTC CAGAAAAAGG GGGGAATGAA AGACCCCACC

 4321 TGTAGGTTTG GCAAGCTACT AGCTTAAGTA ACGCCATTTT GCAAGGCATG GAAAAATACA

 4381 TAACTGAGAA TAGAGAAGTT CAGATCAAGG TCAGGAACAG ATGGAACAGG GTCGATCGAC

 4441 CCTAGAGAAC CATCAGATGT TTCCAGGGTG CCCCAAGGAC CTGAAATGAC CCTGTGCCTT

 4501 ATTTGAACTA ACCAATCAGT TCGCTTCTCG CTTCTGTTCG CGCGCTTCTG CTCCCCGAGC

 4561 TCAATAAAAG AGCCCACAAC CCCTCACTCG GGGCGCCAGT CCTCCGATTG ACTGAGTCGC

 4621 CCGGGTACCC GTGTATCCAA TAAACCCTCT TGCAGTTGCA TCCGACTTGT GGTCTCGCTG

 4681 TTCCTTGGGA GGGTCTCCTC TGAGTGATTG ACTACCCGTC AGCGGGGGTC TTTCATTTGG

 4741 GGGCTCGTCC GGGATCGGGA GACCCCTGCC CAGGGACCAC CGACCCACCA CCGGGAGGTA

 4801 AGCTGGCTGC CTCGCGCGTT TCGGTGATGA CGGTGAAAAC CTCTGACACA TGCAGCTCCC

 4861 GGAGACGGTC ACAGCTTGTC TGTAAGCGGA TGCCGGGAGC AGACAAGCCC GTCAGGGCGC

 4921 GTCAGCGGGT GTTGGCGGGT GTCGGGGCGC AGCCATGACC CAGTCACGTA GCGATAGCGG

 4981 AGTGTAGATC CGGCTGTGGA ATGTGTGTCA GTTAGGGTGT GGAAAGTCCC CAGGCTCCCC

 5041 AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA GCAACCAGGT GTGGAAAGTC

 5101 CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAT

 5161 AGTCCCGCCC CTAACTCCGC CCATCCCGCC CCTAACTCCG CCCAGTTCCG CCCATTCTCC

 5221 GCCCCATGGC TGACTAATTT TTTTTATTTA TGCAGAGGCC GAGGCCGCCT CGGCCTCTGA

 5281 GCTATTCCAG AAGTAGTGAG GAGGCTTTTT TGGAGGCCTA GGCTTTTGCA AAAAGCTTAC

 5341 TGGCTTAACT ATGCGGCATC AGAGCAGATT GTACTGAGAG TGCACCATAT GCGGTGTGAA

 5401 ATACCGCACA GATGCGTAAG GAGAAAATAC CGCATCAGGC GCTCTTCCGC TTCCTCGCTC

 5461 ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG

 5521 GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC

 5581 CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC

 5641 CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA

 5701 CTATAAAGAT ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC

 5761 CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT

 5821 AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG

 5881 CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC

 5941 AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA

 6001 GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT

 6061 AGAAGGACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT

 6121 GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG

 6181 CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG

 6241 TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA

 6301 AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA

 6361 TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG

 6421 ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA

 6481 CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG

 6541 GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT

 6601 GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT

 6661 TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTG CAGGCATCGT GGTGTCACGC

 6721 TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA

 6781 TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT

 6841 AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC

 6901 ATGCCATCCG TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA

 6961 TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA CACGGGATAA TACCGCGCCA

 7021 CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA

 7081 AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT

 7141 TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC

 7201 GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA

 7261 TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT

 7321 TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC

 7381 TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA GGCGTATCAC GAGGCCCTTT

 7441 CGTCTTCAAG AATTAGCTTG GCCATTGCAT ACGTTGTATC CATATCATAA TATGTACATT

 7501 TATATTGGCT CATGTCCAAC ATTACCGCCA TGTTGACATT GATTATTGAC TAGTTATTAA

 7561 TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGT

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