pGBT9 generates a hybrid protein that contains the sequences for the GAL4 DNA-binding domain (DNA-BD; a.a. 1–147). For the construction of a hybrid protein, the gene encoding the protein of interest 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 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 are an intrinsic part of the GAL4 DNA-BD (2). pGBT9 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 TRP1 nutritional marker that allow yeast auxotrophs carrying pGBT9 to grow on limiting synthetic medium lacking Trp.
| 载体基本信息 | |||
| 载体名称 | pGBT9 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 5524 bp | 载体类型 | Yeast Two-Hybrid System |
pGBT9 载体载体图谱
pGBT9 载体载体序列
LOCUS Exported 5524 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 5524)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5524
/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 434..874
/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"
terminator 921..1108
/gene="S. cerevisiae ADH1"
/note="ADH1 terminator"
/note="transcription terminator for the S. cerevisiae
alcohol dehydrogenase 1 (ADH1) gene"
CDS complement(1161..1835)
/codon_start=1
/gene="S. cerevisiae TRP1"
/product="phosphoribosylanthranilate isomerase, required
for tryptophan biosynthesis"
/note="TRP1"
/note="yeast auxotrophic marker"
/translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
KK"
promoter complement(1836..1937)
/gene="S. cerevisiae TRP1"
/note="TRP1 promoter"
primer_bind complement(1952..1968)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 1976..1992
/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(2000..2030)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
rep_origin complement(2354..2942)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3113..3973)
/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(3974..4078)
/gene="bla"
/note="AmpR promoter"
rep_origin 4360..5524
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
