出品公司: | Agilent |
---|---|
载体名称: | pBK-CMV Phagemid vector ;pBK-CMV |
质粒类型: | 真核原核双系统表达载体;克隆载体;噬粒载体 |
高拷贝/低拷贝: | 高拷贝 |
克隆方法: | 多克隆位点,限制性内切酶 |
启动子: | 原核启动子lac;真核启动子CMV |
载体大小: | 4518bp |
5' 测序引物及序列: | -- |
3' 测序引物及序列: | -- |
载体标签: | -- |
载体抗性: | 卡那霉素 |
筛选标记: | 新霉素Neomycin 和卡那霉素Kanamycin |
备注: | 用蓝白斑法筛选原核细胞阳性克隆。 |
产品目录号: | -- |
稳定性: | -- |
组成型/诱导型: | 真核组成型表达,原核IPTG诱导型表达 |
病毒/非病毒: | 非病毒 |
pBK-CMV
价格:3500元
联系方式:I47-825O-882O
买家导航
载体基本信息
载体质粒图谱和多克隆位点信息
载体简介
pBK-CMV Phagemid Vector
The pBK-CMV vector allows you to express proteins in both E. coli and mammalian model systems and saves you valuable time by not having to subclone into an additional vector. In mammalian cells, the expression is driven by the CMV promoter and is therefore constitutively expressed. In E. coli, the lac promoter drives high-level expression in the presence of the inducer IPTG, and allows minimal expression in the absence of inducer.
• Constitutive expression in eukaryotic cells
• Inducible protein expression in prokaryotes
• Rescued from ZAP Express lambda vector
The pBK-CMV phagemid vector is a cloning vector derived from a high-copy-number pUC-based plasmid. This vector allows expression in both eukaryotic and prokaryotic systems. Eukaryotic expression is driven by the cytomegalovirus (CMV) immediate early promoter in the pBK-CMV phagemid vector. Stable clone selection in eukaryotic cells is made possible with G418 by the presence of the neomycin- and kanamycin-resistance gene, which is driven by the SV40 early promoter with thymidine kinase (TK) transcription termination and polyadenylation signals. In the pBK-CMV phagemid vector, prokaryotic expression is driven by the lac promoter, which is repressed in the presence of the LacI protein and is inducible by IPTG. In bacteria expressing the lacZΔM15 mutation and lacI, colonies containing vector without insert will be blue in the presence of X-gal and IPTG. Kanamycin-resistant colonies containing vector with insert will be white and can express the inserted gene as a fusion protein.
Inserts should be cloned within the polylinker cloning sites for prokaryotic expression. For eukaryotic expression one can clone either within the polylinker or between the Nhe I site at the 5´ end of the lac promoter and a polylinker cloning site at the 3´ end. Removing the upstream lac sequences from the eukaryotic transcript by cloning between the Nhe I site and the polylinker sites has been shown to give elevated eukaryotic expression with test inserts.
The pBK–CMV phagemid vector has an extensive polylinker in an SK orientation (the Sac I site is the closest restriction site to the lacZ promoter, and the Kpn I site is the farthest restriction site from the lacZ promoter). The polylinker contains 17 unique restriction enzyme recognition sites, organized with alternating 5´ and 3´ overhangs to allow serial exonuclease III/mung bean nuclease deletions.2 Sites with compatible restriction overhangs, such as Spe I–Xba I and Sal I–Xho I, have been placed on opposite sides of the EcoR I site to allow unidirectional cloning in both the sense and antisense orientations with the ZAP Express vectors and the pBK–CMV phagemid vector. The (–) orientation of the f1 intergenic (IG) region in pBK-CMV allows rescue of antisense single-stranded DNA (ssDNA) relative to thelacZ transcript. This ssDNA can be used for dideoxynucleotide sequencing (Sanger method) or site-specific mutagenesis. Flanking the polylinker are T3 and T7 RNA polymerase promoters that can be used to synthesize RNA in vitro. The choice of promoter used to initiate transcription determines which strand of the DNA insert will be transcribed.
载体序列
LOCUS pBK-CMV 4518 bp DNA SYN DEFINITION pBK-CMV ACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors. FEATURES Location/Qualifiers source 1..4518 /organism="pBK-CMV" /mol_type="other DNA" rep_origin 24..330 /label="f1_origin" misc_feature complement(480..499) /label="EBV_rev_primer" misc_feature complement(748..807) /label="SV40_3_splice" misc_feature complement(823..971) /label="lacZ_a" misc_feature 937..959 /label="M13_pUC_fwd_primer" promoter 952..968 /label="M13_forward20_primer" promoter 975..993 /label="T7_promoter" promoter complement(1139..1158) /label="T3_promoter" misc_feature complement(1192..1214) /label="M13_pUC_rev_primer" promoter complement(1307..1378) /label="CMV_promoter" promoter complement(1330..1894) /label="CMV_immearly_promoter" misc_feature complement(1357..1377) /label="CMV_fwd_primer" misc_feature complement(1543..1830) /label="CAG_enhancer" rep_origin complement(1995..2614) /label="pBR322_origin" terminator complement(2762..3031) /label="TK_PA_terminator" CDS 3155..3691 /label="ORF frame 2" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" CDS complement(3206..4000) /label="ORF frame 3" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" gene complement(3209..3988) /label="NeoR/KanR (variant)" /gene="NeoR/KanR (variant)" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" promoter complement(4083..4351) /label="SV40_promoter" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" misc_feature complement(4103..4122) /label="SV40pro_F_primer" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" rep_origin complement(4107..4184) /label="SV40_origin" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" misc_feature 4154..4369 /label="SV40_enhancer" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" misc_feature 4349..4369 /label="pBABE_3_primer" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" promoter complement(4435..4463) /label="AmpR_promoter" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" ORIGIN 1 CACCTGACGC GCCCTGTAGC GGCGCATTAA GCGCGGCGGG TGTGGTGGTT ACGCGCAGCG 61 TGACCGCTAC ACTTGCCAGC GCCCTAGCGC CCGCTCCTTT CGCTTTCTTC CCTTCCTTTC 121 TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG GGGGCTCCCT TTAGGGTTCC 181 GATTTAGTGC TTTACGGCAC CTCGACCCCA AAAAACTTGA TTAGGGTGAT GGTTCACGTA 241 GTGGGCCATC GCCCTGATAG ACGGTTTTTC GCCCTTTGAC GTTGGAGTCC ACGTTCTTTA 301 ATAGTGGACT CTTGTTCCAA ACTGGAACAA CACTCAACCC TATCTCGGTC TATTCTTTTG 361 ATTTATAAGG GATTTTGCCG ATTTCGGCCT ATTGGTTAAA AAATGAGCTG ATTTAACAAA 421 AATTTAACGC GAATTTTAAC AAAATATTAA CGCTTACAAT TTACGCGTTA AGATACATTG 481 ATGAGTTTGG ACAAACCACA ACTAGAATGC AGTGAAAAAA ATGCTTTATT TGTGAAATTT 541 GTGATGCTAT TGCTTTATTT GTAACCATTA TAAGCTGCAA TAAACAAGTT AACAACAACA 601 ATTGCATTCA TTTTATGTTT CAGGTTCAGG GGGAGGTGTG GGAGGTTTTT TAAAGCAAGT 661 AAAACCTCTA CAAATGTGGT ATGGCTGATT ATGATCATGA ACAGACTGTG AGGACTGAGG 721 GGCCTGAAAT GAGCCTTGGG ACTGTGAATC TAAAATACAC AAACAATTAG AATCAGTAGT 781 TTAACACATT ATACACTTAA AAATTGGATC TCCATTCGCC ATTCAGGCTG CGCAACTGTT 841 GGGAAGGGCG ATCGGTGCGG GCCTCTTCGC TATTACGCCA GCTGGCGAAA GGGGGATGTG 901 CTGCAAGGCG ATTAAGTTGG GTAACGCCAG GGTTTTCCCA GTCACGACGT TGTAAAACGA 961 CGGCCAGTGA ATTGTAATAC GACTCACTAT AGGGCGAATT GGGTACACTT ACCTGGTACC 1021 CCACCCGGGT GGAAAATCGA TGGGCCCGCG GCCGCTCTAG AAGTACTCTC GAGAAGCTTT 1081 TTGAATTCTT TGGATCCACT AGTGTCGACC TGCAGGCGCG CGAGCTCCAG CTTTTGTTCC 1141 CTTTAGTGAG GGTTAATTTC GAGCTTGGCG TAATCAAGGT CATAGCTGTT TCCTGTGTGA 1201 AATTGTTATC CGCTCACAAT TCCACACAAT ATACGAGCCG GAAGTATAAA GTGTAAAGCC 1261 TGGGGTGCCT AATGAGTGAG CTAACTCACA GTAATTGCGG CTAGCGGATC TGACGGTTCA 1321 CTAAACCAGC TCTGCTTATA TAGACCTCCC ACCGTACACG CCTACCGCCC ATTTGCGTCA 1381 ATGGGGCGGA GTTGTTACGA CATTTTGGAA AGTCCCGTTG ATTTTGGTGC CAAAACAAAC 1441 TCCCATTGAC GTCAATGGGG TGGAGACTTG GAAATCCCCG TGAGTCAAAC CGCTATCCAC 1501 GCCCATTGAT GTACTGCCAA AACCGCATCA CCATGGTAAT AGCGATGACT AATACGTAGA 1561 TGTACTGCCA AGTAGGAAAG TCCCATAAGG TCATGTACTG GGCATAATGC CAGGCGGGCC 1621 ATTTACCGTC ATTGACGTCA ATAGGGGGCG TACTTGGCAT ATGATACACT TGATGTACTG 1681 CCAAGTGGGC AGTTTACCGT AAATACTCCA CCCATTGACG TCAATGGAAA GTCCCTATTG 1741 GCGTTACTAT GGGAACATAC GTCATTATTG ACGTCAATGG GCGGGGGTCG TTGGGCGGTC 1801 AGCCAGGCGG GCCATTTACC GTAAGTTATG TAACGCGGAA CTCCATATAT GGGCTATGAA 1861 CTAATGACCC CGTAATTGAT TACTATTAAT AACTAATGCA TGGCGGTAAT ACGGTTATCC 1921 ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG 1981 AACCGTAAAA AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT 2041 CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG 2101 GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA 2161 TACCTGTCCG CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG 2221 TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT 2281 CAGCCCGACC GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC 2341 GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC 2401 GGTGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT 2461 GGTATCTGCG CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC 2521 GGCAAACAAA CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC 2581 AGAAAAAAAG GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG 2641 AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG 2701 ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAACCTGAG 2761 GCTATGGCAG GGCCTGCCGC CCCGACGTTG GCTGCGAGCC CTGGGCCTTC ACCCGAACTT 2821 GGGGGGTGGG GTGGGGAAAA GGAAGAAACG CGGGCGTATT GGCCCCAATG GGGTCTCGGT 2881 GGGGTATCGA CAGAGTGCCA GCCCTGGGAC CGAACCCCGC GTTTATGAAC AAACGACCCA 2941 ACACCGTGCG TTTTATTCTG TCTTTTTATT GCCGTCATAG CGCGGGTTCC TTCCGGTATT 3001 GTCTCCTTCC GTGTTTCAGT TAGCCTCCCC CTAGGGTGGG CGAAGAACTC CAGCATGAGA 3061 TCCCCGCGCT GGAGGATCAT CCAGCCGGCG TCCCGGAAAA CGATTCCGAA GCCCAACCTT 3121 TCATAGAAGG CGGCGGTGGA ATCGAAATCT CGTGATGGCA GGTTGGGCGT CGCTTGGTCG 3181 GTCATTTCGA ACCCCAGAGT CCCGCTCAGA AGAACTCGTC AAGAAGGCGA TAGAAGGCGA 3241 TGCGCTGCGA ATCGGGAGCG GCGATACCGT AAAGCACGAG GAAGCGGTCA GCCCATTCGC 3301 CGCCAAGCTC TTCAGCAATA TCACGGGTAG CCAACGCTAT GTCCTGATAG CGGTCCGCCA 3361 CACCCAGCCG GCCACAGTCG ATGAATCCAG AAAAGCGGCC ATTTTCCACC ATGATATTCG 3421 GCAAGCAGGC ATCGCCATGG GTCACGACGA GATCCTCGCC GTCGGGCATG CTCGCCTTGA 3481 GCCTGGCGAA CAGTTCGGCT GGCGCGAGCC CCTGATGCTC TTCGTCCAGA TCATCCTGAT 3541 CGACAAGACC GGCTTCCATC CGAGTACGTG CTCGCTCGAT GCGATGTTTC GCTTGGTGGT 3601 CGAATGGGCA GGTAGCCGGA TCAAGCGTAT GCAGCCGCCG CATTGCATCA GCCATGATGG 3661 ATACTTTCTC GGCAGGAGCA AGGTGAGATG ACAGGAGATC CTGCCCCGGC ACTTCGCCCA 3721 ATAGCAGCCA GTCCCTTCCC GCTTCAGTGA CAACGTCGAG CACAGCTGCG CAAGGAACGC 3781 CCGTCGTGGC CAGCCACGAT AGCCGCGCTG CCTCGTCTTG CAGTTCATTC AGGGCACCGG 3841 ACAGGTCGGT CTTGACAAAA AGAACCGGGC GCCCCTGCGC TGACAGCCGG AACACGGCGG 3901 CATCAGAGCA GCCGATTGTC TGTTGTGCCC AGTCATAGCC GAATAGCCTC TCCACCCAAG 3961 CGGCCGGAGA ACCTGCGTGC AATCCATCTT GTTCAATCAT GCGAAACGAT CCTCATCCTG 4021 TCTCTTGATC GATCTTTGCA AAAGCCTAGG CCTCCAAAAA AGCCTCCTCA CTACTTCTGG 4081 AATAGCTCAG AGGCCGAGGC GGCCTCGGCC TCTGCATAAA TAAAAAAAAT TAGTCAGCCA 4141 TGGGGCGGAG AATGGGCGGA ACTGGGCGGA GTTAGGGGCG GGATGGGCGG AGTTAGGGGC 4201 GGGACTATGG TTGCTGACTA ATTGAGATGC ATGCTTTGCA TACTTCTGCC TGCTGGGGAG 4261 CCTGGGGACT TTCCACACCT GGTTGCTGAC TAATTGAGAT GCATGCTTTG CATACTTCTG 4321 CCTGCTGGGG AGCCTGGGGA CTTTCCACAC CCTAACTGAC ACACATTCCA CAGCTGGTTC 4381 TTTCCGCCTC AGGACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC 4441 TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA 4501 CATTTCCCCG AAAAGTGC //
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