pACT2 generates a fusion of the GAL4 AD (amino acids 768–881), an HA epitope tag, and a protein of interest (or protein encoded by a cDNA in a fusion library) cloned into the MCS in the correct orientation and reading frame. pACT2, which is derived from pACT (1), contains a unique EcoR I site in the MCS. The hybrid protein is expressed at high levels in yeast host cells from the constitutive ADH1 promoter (P); transcription is terminated at the ADH1 transcription termination signal (T). The protein is targeted to the yeast nucleus by the nuclear localization sequence from SV40 T-antigen which has been cloned into the 5’ end of the GAL4 AD sequence (2). pACT2 is a shuttle vector that replicates autonomously in both E. coli and S. cerevisiae and carries the bla gene, which confers ampicillin resistance in E. coli. pACT2 also contains the LEU2 nutritional gene that allows yeast auxotrophs to grow on limiting synthetic media. Transformants with AD/library plasmids can be selected by complementation by the LEU2 gene by using an E. coli strain that carries a leuB mutation (e.g., HB101).
| 载体基本信息 | |||
| 载体名称 | pACT2 AD | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 8117 bp | 载体类型 | Yeast Two-Hybrid System |
| 启动子 | ADH1 | 克隆方法 | Enzyme digestion and ligation |
pACT2 AD 载体载体图谱
pACT2 AD 载体载体序列
LOCUS Exported 8117 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 8117)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8117
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 238..1580
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
protein_bind 570..617
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FRT"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
promoter 2057..2461
/gene="S. cerevisiae LEU2"
/note="LEU2 promoter"
CDS 2474..3568
/codon_start=1
/gene="S. cerevisiae LEU2"
/product="3-isopropylmalate dehydrogenase, required for
leucine biosynthesis"
/note="LEU2"
/note="yeast auxotrophic marker"
/translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
DLGGSNSTTEVGDAVAEEVKKILA"
protein_bind complement(4186..4219)
/bound_moiety="Cre recombinase"
/note="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
protein_bind complement(4263..4296)
/bound_moiety="Cre recombinase"
/note="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
protein_bind complement(4368..4401)
/bound_moiety="Cre recombinase"
/note="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
terminator 4419..4606
/gene="S. cerevisiae ADH1"
/note="ADH1 terminator"
/note="transcription terminator for the S. cerevisiae
alcohol dehydrogenase 1 (ADH1) gene"
CDS complement(5042..5068)
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/note="HA"
/translation="YPYDVPDYA"
CDS complement(5081..5422)
/codon_start=1
/gene="Saccharomyces cerevisiae GAL4 (truncated)"
/product="activation domain of the GAL4 transcriptional
activator"
/note="GAL4 activation domain"
/translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
EDTPPNPKKE"
CDS complement(5438..5458)
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
promoter complement(5504..5900)
/gene="S. cerevisiae ADH1"
/note="ADH1 promoter"
/note="promoter for alcohol dehydrogenase 1"
misc_feature 6042..6182
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(6368..6956)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7127..7987)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(7988..8092)
/gene="bla"
/note="AmpR promoter"
