The pACT Vector is a high-copy plasmid in which the human cytomegalovirus (CMV) immediate early promoter drives expression of the herpes virus VP16 activation domain (amino acids 411–456). A chimeric intron is located 5´ of the gene segment, and a multiple cloning region is located 3´ of the gene segment for insertion of cDNA clones of interest (Figures 2 and 3). The presence of this chimeric intron, in context with the CMV promoter and polyadenylation signal, can result in increased protein expression of cDNA genes linked to these elements (6). The stop codons and SV40 late polyadenylation region, demonstrated to be efficient for mRNA production (7), are at the 3´ end of the fusion gene. The fusion gene region is flanked by T7 and T3 RNA polymerase promoters for the synthesis of sense and antisense RNA products. The T7 promoter allows the construct to be translated using the TNT T7 Quick Coupled Transcription/Translation System (Cat.# L1170). Also located on this vector is the neomycin phosphotransferase gene (from Tn5) driven by the SV40 early promoter and followed by a synthetic polyadenylation cassette. This gene confers resistance to the neomycin analog, G418 (Geneticin; 8). The plasmid backbone contains an f1 origin of replication for the production of single-stranded DNA (ssDNA) and the β-lactamase (Ampr) gene for selection of the vector DNA in E. coli.
| 载体基本信息 | |||
| 载体名称 | pACT | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 5566 bp | 载体类型 | Mammalian Two-Hybrid System |
| 筛选标记 | Neomycin | 启动子 | CMV |
pACT 载体载体图谱
pACT 载体载体序列
LOCUS Exported 5566 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 5566)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5566
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 138..517
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 518..729
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
intron 890..1022
/note="chimeric intron"
/note="chimera between introns from human beta-globin and
immunoglobulin heavy chain genes"
promoter 1067..1085
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1149..1169
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
promoter complement(1403..1420)
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
polyA_signal 1438..1559
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 1693..2148
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2163..2520
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2371..2506
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2571..3365
/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
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3429..3477
/note="synthetic polyadenylation signal"
promoter 3769..3873
/gene="bla"
/note="AmpR promoter"
CDS 3874..4734
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 4905..5493
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
