The pCMV-BD and pCMV-AD vectors are designed for the construction and expression of gene fusions with the GAL4 DNA binding domain and NF-κB transcriptional activation domain, respectively. The pCMV-BD vector contains DNA encoding amino acids 1–147 of the GAL4 gene (DNA binding domain)2 and unique 3′ cloning sites. It is used for the construction of bait plasmids containing a DNA insert encoding a bait protein. The pCMV-AD vector contains DNA encoding the nuclear localization sequence (NLS) from SV40 large T-antigen (PKKKRKV), amino acids 364–550 of the mouse NF-κB gene (transcriptional activation domain)3 and unique 3′ cloning sites. It is used for constructing a target plasmid containing a DNA insert encoding the interacting proteins. Both pCMV-BD and pCMV-AD contain the pUC origin for replication in E. coli. The pCMV-BD vector contains the kanamycin-resistance gene and the pCMV-AD vector contains the ampicillin-resistance gene. The constitutively-active cytomegalovirus (CMV) promoter governs the expression of the bait and target proteins in the pCMV-BD and pCMV-AD vectors. The SV40 poly (A) provide the signals necessary for transcriptional termination and polyadenylation of the bait and target genes in mammalian cells.
| 载体基本信息 | |||
| 载体名称 | pCMV-BD | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 4730 bp | 载体类型 | Mammalian Two-Hybrid System |
| 筛选标记 | Neomycin | 拷贝数 | High copy number |
| 启动子 | CMV | 载体标签 | Gal4-DBD |
pCMV-BD 载体载体图谱
pCMV-BD 载体载体序列
LOCUS Exported 4730 bp ds-DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4730)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4730
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 67..370
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 371..574
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 620..638
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
CDS 675..1115
/codon_start=1
/gene="Saccharomyces cerevisiae GAL4 (truncated)"
/product="DNA binding domain of the GAL4 transcriptional
activator"
/note="GAL4 DNA binding domain"
/translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
promoter complement(1232..1250)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
polyA_signal 1524..1645
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(1652..2107)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2134..2238
/gene="bla"
/note="AmpR promoter"
promoter 2240..2597
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2448..2583
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2632..3426
/codon_start=1
/gene="aph(3)-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3658..3705
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4034..4622
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
