pQCXIN载体简介pQCXIN Retroviral Vector is a bicistronic expression vector designed to express a target gene along with the neomycin selection marker (1). Upon transfection into a packaging cell line, this vector can transiently express, or integrate and stably express a viral genomic transcript containing the CMV immediate early promoter, gene of interest, IRES and the neomycin resistance gene (Neor). The gene of interest and the neomycin resistance gene are co-translated, via the internal ribosome entry site (IRES), from a bicistronic message in mammalian cells (2, 3).This vector incorporates unique features including: optimization to remove promoter interference and self-inactivation. The hybrid 5' LTR consists of the cytomegalovirus (CMV) type I enhancer and the mouse sarcoma virus (MSV) promoter. This construct drives high levels of transcription in HEK 293-based packaging cell lines due, in part, to the presence of adenoviral E1A (4, 5, 6, 7) 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, resulting in inactivation of the 5' LTR CMV enhancer sequences. This may reduce the phenomenon known as promoter interference (8) and allow more efficient expression.Also included in the viral genomic transcript are the necessary viral RNA processing elements including the LTRs, packaging signal (Psi+), and tRNA primer binding site. pQCXIN also contains a bacterial origin of replication and E. coli Ampr gene for propagation and selection in bacteria.pQCXIN载体的使用pQCXIN is designed to deliver and express a gene along with the neomycin resistance marker from a bicistronic message. The design is optimized to produce high titers via the PCMV IE in the packaging cell line. The bicistronic transcript makes it unnecessary to screen the transformants since the neomycin resistance is expressed in concert with the DNA inserted into the multiple cloning site.Once transfected into the packaging cell line (such as the RetroPack™ PT67 Cell Line (Cat. No.631510) AmphoPack293, EcoPack2-293, or Pantropic System), RNA from the vector is packaged into infectious, replication-incompetent retroviral particles since pQCXIN lacks structural genes (gag, pol, and env) necessary for particle formation and replication; however, these genes are stably integrated as part of the packaging cell genome. Once a high titer clone is selected, 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 introductioin and integration of the structural genes into the packaging cell line minimizes the chances of producing replication-competent virus due to recomination events during cell proliferation.
LOCUS pQCXIN 7381 bp DNA SYNDEFINITION pQCXINACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors.FEATURES Location/Qualifiers source 1..7381 /organism="pQCXIN" /mol_type="other DNA" promoter 2..506 /label="CMV_immearly_promoter" misc_feature 6..688 /label="5_LTR" misc_feature 9..296 /label="CAG_enhancer" misc_feature 463..483 /label="CMV_fwd_primer" misc_feature 758..1567 /label="psi_plus_pack" misc_feature 1154..1561 /label="gag" misc_feature 1497..1519 /label="pLXSN_5_primer" misc_feature 1497..1519 /label="MSCV_primer" misc_feature 1535..1551 /label="pBABE_5_primer" misc_feature 1608..1895 /label="CAG_enhancer" promoter 1613..2105 /label="CMV_immearly_promoter" misc_feature 2062..2082 /label="CMV_fwd_primer" promoter 2063..2132 /label="CMV_promoter" promoter 2075..2194 /label="CMV2_promoter" misc_feature 2106..2125 /label="pCEP_fwd_primer" misc_feature 2108..2132 /label="LNCX_primer" misc_feature 2289..2851 /label="IRES" CDS 2876..3670 /label="ORF frame 2" /translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" gene 2891..3667 /label="NeoR/KanR" /gene="NeoR/KanR" /translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" misc_feature complement(4615..4637) /label="pGEX_3_primer" /translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" misc_feature complement(4773..4793) /label="pBABE_3_primer" /translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" misc_feature complement(4779..4994) /label="SV40_enhancer" /translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" promoter 4791..5059 /label="SV40_promoter" /translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" rep_origin 4958..5035 /label="SV40_origin" /translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" misc_feature 5020..5039 /label="SV40pro_F_primer" /translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" gene complement(6159..7019) /label="Ampicillin" /gene="Ampicillin" /translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" CDS complement(6159..7019) /label="ORF frame 3" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*" promoter complement(7061..7089) /label="AmpR_promoter" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*" promoter 7311..506 /label="CMV_immearly_promoter" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*" misc_feature 7375..688 /label="5_LTR" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*"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 tgggggatcg atcctctaga gtccgttaca taacttacgg 1621 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt 1681 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac 1741 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg 1801 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact 1861 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt 1921 ggcagtacat caatgggcgt gaatagcggt ttgactcacg gggatttcca agtctccacc 1981 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc