| 出品公司: | ZYbscience |
|---|---|
| 载体名称: | pCDF1-MCS2-EF1-copGFP |
| 质粒类型: | 慢病毒表达载体；cDNA表达载体 |
| 克隆方法: | 多克隆位点，限制性内切酶 |
| 启动子: | CMV |
| 载体大小: | 6771bp |
| 5测序引物及序列: | LNCX-FAGCTCGTTTAGTGAACCGTCAGATC |
| 3测序引物及序列: | EF1a-R |
| 载体标签: | 无 |
| 载体抗性: | 氨苄青霉素（Ampicillin） |
| 筛选标记: | GFP |
| 克隆菌株: | stbl3或者E.coli(RecA-)：OmniMAX2 |
| 宿主细胞（系）: | 常用细胞系(e.g.HeLa,HEK293,HT1080,H1299) |
| 备注: | pCDF1-MCS2-EF1-copGFP慢病毒表达载体是基于FIV的慢病毒载体； 用于cDNA表达和克隆；高效转染细胞，建立稳定细胞系，表达水平高； |
| 产品目录号: | ZY111B-1 |
| 稳定性: | 稳表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 慢病毒（FIV) |
人凝血酶(Thrombin)ELISA试剂盒说明书
| 货号： | ZY111B-1 |
| 供应商： | 泽叶生物 |
| 数量： | 20 |
| 英文名： | pCDF1-MCS2-EF1-copGFP |
| 保质期： | 三年 |
| 保存条件： | -20℃低温保存 |
载体基本信息
载体质粒图谱和多克隆位点信息
载体简介
背景简介：A. Purpose of this ManualThis manual provides details and information necessary to generate expression constructs of your gene of interest in the pCDF lentivectors. Specifically, it provides critical instructions on amplification and cloning the cDNA into the pCDF Vectors, and verifying final expression constructs. This manual does not include information on packaging the pCDF expression constructs into pseudotyped viral particles or transducing your target cells of choice with these particles. B. Advantages of the Lentivector Expression System Lentiviral expression vectors are the most effective vehicles for delivering and expression of a gene of interest to almost any mammalian cell—including non-dividing cells and model organisms (C.A. Machida, 2003; M. Federico, 2003; W. C. Heiser, 2004). As with standard plasmid vectors, it is possible to introduce lentivector expression constructs in plasmid form into the cells with low-tomedium efficiency using conventional transfection protocols.However, by packaging the lentivector construct into viral particles, you can obtain highly efficient transduction of expression constructs—even with the most difficult to transfect cells, such as primary, stem, and differentiated cells. The expression construct transduced in target cells is integrated into genomic DNA and provides stable, long-term expression of the target gene.The lentiviral cDNA expression system consists of three main components:(1) The lentiviral expression vector (e.g., pCDF1-MCS2-EF1-Puro)(2) The lentiviral packaging plasmids (e.g., pPACKF1 Packaging Plasmid mix)(3) A pseudoviral particle producer cell line (e.g., 293TN cells) The expression lentivector contains the genetic elements responsible for packaging, transduction, stable integration of the viral expression construct into genomic DNA, and expression of the target gene sequence. The packaging vector provides all the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant viral particles. To produce a high titer of viral particles, expression and packaging vectors are transiently cotransfected into producer mammalian cells (e.g., HEK 293 cells). For a detailed description of SBI’s Lentivector expression system, please refer to the Lentivector Expression Systems user manual.SBI’s novel pCDF Vectors are derived from feline immunodeficiency virus (FIV; Poeschla, 2003; for Safety Guidelines when working with these vectors, see section G). These pCDF Vectors, developed at SBI, are self-inactivating as a result of a deletion in the U3 region of 3’ ΔLTR (see Appendix for Vector Features). Upon integration into the genome, the 5’ LTR promoter is inactivated, which prevents formation of replication-competent viral particles.When expressed, the hybrid CMV/FIV 5’ LTR drives high level transcription of the viral construct and produces a transcript that contains all the necessary functional elements (i.e., Psi, RRE, and cPPT) for efficient packaging. When this construct is expressed in HEK 293 cells that also express viral coat proteins (i.e., a packaging cell line), the pCDF transcripts are efficiently packaged into pseudoviral particles. After isolation, these pseudoviral particles containing the RNA version of the pCDF expression cassette can be efficiently transduced into any mammalian target cells. Following transduction into the target cells, this expression cassette is reverse transcribed and integrated into the genome of the target cell. The pCDF Vectors also contain a bacterial origin of replication and ampicillin resistance (AmpR) gene for propagation and selection in E.coli. The pCDF1-MCS2-EF1-Puro Vector (Cat. # CD110B-1) contains a puromycin resistance gene, under the control of a constitutive EF1 promoter and a WPRE regulatory element, to enable selection of target cells stably expressing the cDNA template. The pCDF1-MCS2-EF1-copGFP Vector (Cat. # CD111B-1) contains a copGFP gene under the control of a EF1 promoter and WPRE element. CopGFP is a novel fluorescent protein ,derived from copepod plankton (Panalina sp.), which is similar to EGFP but has a brighter color This gene serves as a reporter for the transfected or transduced cells. pCDF Cloning and Expression LentivectorsThe FIV derived pCDF vectors contain the following features: CMV promoter—promotes a high level of expression of your gene of interest in a wide variety of cell lines. Multiple Cloning Site (MCS)—for cloning the gene of interest in MCS located downstream of CMV promoter. WPRE element—enhances stability and translation of the CMVdriven transcripts. SV40 polyadenylation signal—enables efficient termination of transcription and processing of recombinant transcripts. Optional second expression cassette—provides expression of puromycin resistance gene or copGFP reporter under control of constitutive elongation factor 1 (EF1) promoter for selection or FACS analysis of transduced cells. Hybrid CMV-5LTR promoter—provides a high level of expression of the full-length viral transcript in producer 293 cells. Genetic elements (cPPT, GAG, LTRs)—necessary for packaging, transducing, and stably integrating the viral expression construct into genomic DNA. SV40 origin—for stable propagation of the pCDF plasmid in mammalian cells. pUC origin—for high copy replication and maintenance of the plasmid in E.coli cells. Ampicillin resistance gene—for selection in E.coli cells.
载体序列
LOCUS pCDF1-MCS2-EF1-copGFP 6771 bp DNA SYNDEFINITION pCDF1-MCS2-EF1-copGFPACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors.FEATURES Location/Qualifiers source 1..6771 /organism="pCDF1-MCS2-EF1-copGFP" /mol_type="other DNA" misc_feature 287..307 /label="CMV_fwd_primer" misc_feature 1165..1180 /label="cPPT" misc_feature 1633..1653 /label="CMV_fwd_primer" promoter 1634..1703 /label="CMV_promoter" misc_feature 1677..1696 /label="pCEP_fwd_primer" misc_feature 1679..1703 /label="LNCX_primer" CDS 2366..3124 /label="ORF frame 2" /translation="MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNK MKSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGG VLHVSFSYRYEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGS FARTFSLRDGGYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEY QHAFKTPIAFARSRAQSSNSAVDGTAGPGSTGSR*" misc_feature 3133..3708 /label="WPRE" /translation="MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNK MKSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGG VLHVSFSYRYEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGS FARTFSLRDGGYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEY QHAFKTPIAFARSRAQSSNSAVDGTAGPGSTGSR*" CDS 3221..3961 /label="ORF frame 2" /translation="MPLYHAIASRMAFIFSSLYKSWLLSLYEELWPVVRQRGVVCTVF ADATPTGWGIATTCQLLSGTFAFPLPIATAELIAACLARCWTGARLLGTDNSVVLSGK SSSFPWLLACVATWILRGTSFCYVPSALNPADLPSRGLLPALRPLPRLRLRPQTSRIS LWAASPPVPMTELEDRFRKLFGTTSTTGDSTVDSEDEPPKKEKRVDWDEYWNPEIDSF QCFVKLRGVSLLRTFEFSLEAPTDTINI*" CDS 3234..3731 /label="ORF frame 3" /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP PRRYR*" misc_feature 3818..3843 /label="U3PPT" /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP PRRYR*" misc_feature 3820..3843 /label="U3PPT" /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP PRRYR*" terminator 4058..4177 /label="SV40_PA_terminator" /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP PRRYR*" misc_feature 4146..4165 /label="EBV_rev_primer" /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP PRRYR*" rep_origin 4195..4272 /label="SV40_origin" /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP PRRYR*" misc_feature 4257..4276 /label="SV40pro_F_primer" /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP PRRYR*" promoter complement(4364..4382) /label="M13_reverse_primer" /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP PRRYR*" misc_feature complement(4381..4403) /label="M13_pUC_rev_primer" /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP PRRYR*" promoter complement(4417..4446) /label="lac_promoter" /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP PRRYR*" gene complement(5529..6389) /label="Ampicillin" /gene="Ampicillin" /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP PRRYR*" CDS complement(5529..6389) /label="ORF frame 2" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*" promoter complement(6431..6459) /label="AmpR_promoter" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*" misc_feature complement(6618..6640) /label="pGEX_3_primer" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*"ORIGIN 1 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat 61 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc 121 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac 181 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa 241 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt 301 aggcgtgtac ggtgggaggt ctatataagc agagcttgtg aaacttcgag gagtctcttt 361 gttgaggact tttgagttct cccttgaggc tcccacagat acaataaata tttgagattg 421 aaccctgtcg agtatctgtg taatcttttt tacctgtgag gtctcggaat ccgggccgag 481 aacttcgcag ttggcgcccg aacagggact tgattgagag tgattgagga agtgaagcta 541 gagcaataga aagctgttaa gcagaactcc tgctgaccta aatagggaag cagtagcaga 601 cgctgctaac agtgagtatc tctagtgaag cagactcgag ctcataatca agtcattgtt 661 taaaggccca gataaattac atctggtgac tcttcgcgga ccttcaagcc aggagattcg 721 ccgagggaca gtcaacaagg taggagagat tctacagcaa catggggaat ggacaggggc 781 gagattggaa aatggccatt aagagatgta gtaatgttgc tgtaggagta ggggggaaga 841 gtaaaaaatt tggagaaggg aatttcagat gggccattag aatggctaat gtatctacag 901 gacgagaacc tggtgatata ccagagactt tagatcaact aaggttggtt atttgcgatt 961 tacaagaaag aagagaaaaa tttggatcta gcaaagaaat tgatatggca attcctgcat 1021 tgaggagaaa tggtaggcaa tgtggcatgt ctgaaaaaga ggaggaatga tgaagtatct 1081 cagacttatt ttataaggga gatactgtgc tgagttcttc cctttgagga aggtatgtca 1141 tatcctagac atagtctcaa ttttaaaaga agaggtagga taggagggat ggccccttat 1201 gaattattag cacaacaaga atccttaaga atacaagatt atttttctgc aataccacaa 1261 aaattgcaag cacagtggat ttattataaa gatcaaaaag ataagaaatg gaaaggacca 1321 atgagagtag aatactgggg acagggatca gtattattaa aggatgaaga gaagggatat 1381 tttcttataa tcgatactag tattatgccc agtacatgac cttatgggac tttcctactt 1441 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca 1501 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 1561 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact 1621 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag 1681 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata 1741 gaagattcta gagcccgggc gcgccggatc cagatcttaa ttaatttaaa tgaattcgcg 1801 gccgcgaagg atctgcgatc gctccggtgc ccgtcagtgg gcagagcgca catcgcccac 1861 agtccccgag aagttggggg gaggggtcgg caattgaacg ggtgcctaga gaaggtggcg 1921 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg 1981 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc 2041 cagaacacag ctgaagcttc gaggggctcg catctctcct tcacgcgccc gccgccctac 2101 ctgaggccgc catccacgcc ggttgagtcg cgttctgccg cctcccgcct gtggtgcctc 2161 ctgaactgcg tccgccgtct aggtaagttt aaagctcagg tcgagaccgg gcctttgtcc 2221 ggcgctccct tggagcctac ctagactcag ccggctctcc acgctttgcc tgaccctgct 2281 tgctcaactc tacgtctttg tttcgttttc tgttctgcgc cgttacagat ccaagctgtg 2341 accggcgcct acgctagacg ccaccatgga gagcgacgag agcggcctgc ccgccatgga 2401 gatcgagtgc cgcatcaccg gcaccctgaa cggcgtggag ttcgagctgg tgggcggcgg 2461 agagggcacc cccaagcagg gccgcatgac caacaagatg aagagcacca aaggcgccct 2521 gaccttcagc ccctacctgc tgagccacgt gatgggctac ggcttctacc acttcggcac 2581 ctaccccagc ggctacgaga accccttcct gcacgccatc aacaacggcg gctacaccaa 2641 cacccgcatc gagaagtacg aggacggcgg cgtgctgcac gtgagcttca gctaccgcta 2701 cgaggccggc cgcgtgatcg gcgacttcaa ggtggtgggc accggcttcc ccgaggacag 2761 cgtgatcttc accgacaaga tcatccgcag caacgccacc gtggagcacc tgcaccccat 2821 gggcgataac gtgctggtgg gcagcttcgc ccgcaccttc agcctgcgcg acggcggcta 2881 ctacagcttc gtggtggaca gccacatgca cttcaagagc gccatccacc ccagcatcct 2941 gcagaacggg ggccccatgt tcgccttccg ccgcgtggag gagctgcaca gcaacaccga 3001 gctgggcatc gtggagtacc agcacgcctt caagaccccc atcgccttcg ccagatcccg 3061 cgctcagtcg tccaattctg ccgtggacgg caccgccgga cccggctcca ccggatctcg 3121 ctaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa 3181 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat 3241 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta 3301 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc 3361 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt 3421 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg 3481 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc 3541 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc 3601 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct 3661 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc 3721 ggtaccgatg acagagttag aagatcgctt caggaagcta tttggcacga cttctacaac 3781 gggagacagc acagtagatt ctgaagatga acctcctaaa aaagaaaaaa gggtggactg 3841 ggatgagtat tggaaccctg aaatcgatag cttccagtgc tttgtgaaac ttcgaggagt 3901 ctctttgttg aggacttttg agttctccct tgaggctccc acagatacaa taaatatttg 3961 agattgaacc ctgtcgagta tctgtgtaat cttttttacc tgtgaggtct cggaatccgg
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发布于 ： 2018-02-04
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