| 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 |
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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