pGAD424 generates a hybrid protein that contains the sequences for the GAL4 activation domain (AD; a.a. 768–881) (1). pGAD424 has unique restriction sites located in the MCS region at the 3- end of the open reading frame for the GAL4 AD sequence. For the construction of a hybrid protein, the gene encoding the protein of interest (or a collection of cDNAs) is ligated into the MCS in the correct orientation and in the correct reading frame such that a fusion protein is generated. The fusion protein is expressed at high levels in yeast host cells from the constitutive ADH1 promoter; transcription is terminated at the ADH1 transcription termination signal. The hybrid protein is targeted to the yeast nucleus by nuclear localization sequences that have been added to the AD sequence from a heterologous source (2). pGAD424 is a shuttle vector that replicates autonomously in both E. coli and S. cerevisiae. It carries the bla gene (for ampicillin resistance in E. coli) and the LEU2 nutritional marker that allow yeast auxotrophs carrying pGAD424 to grow on limiting synthetic medium lacking Leu.
| 载体基本信息 | |||
| 载体名称 | pGAD424 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 6659 bp | 载体类型 | Yeast one hybrid systems |
| 启动子 | ADH1 | ||
pGAD424 载体载体图谱
pGAD424 载体载体序列
LOCUS Exported 6659 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 6659)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..6659
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 5..406
/gene="S. cerevisiae ADH1"
/note="ADH1 promoter"
/note="promoter for alcohol dehydrogenase 1"
CDS 452..472
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 488..829
/codon_start=1
/gene="Saccharomyces cerevisiae GAL4 (truncated)"
/product="activation domain of the GAL4 transcriptional
activator"
/note="GAL4 activation domain"
/translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
EDTPPNPKKE"
terminator 1241..1428
/gene="S. cerevisiae ADH1"
/note="ADH1 terminator"
/note="transcription terminator for the S. cerevisiae
alcohol dehydrogenase 1 (ADH1) gene"
CDS complement(1546..2640)
/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"
promoter complement(2641..3045)
/gene="S. cerevisiae LEU2"
/note="LEU2 promoter"
primer_bind complement(3087..3103)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3111..3127
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(3135..3165)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
rep_origin complement(3489..4077)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4248..5108)
/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"
promoter complement(5109..5213)
/gene="bla"
/note="AmpR promoter"
rep_origin 5495..6659
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
