pHIS2 is a reporter vector that can be used in yeast one-hybrid assays to identify and characterize DNA-binding proteins. The vector was specifically designed for use with the BD Matchmaker One- Hybrid Library Construction & Screening Kit (#K1617-1). It contains a HIS3 nutritional reporter gene, located downstream of a multiple cloning site (MCS) and the minimal promoter of the HIS3 locus (PminHIS3). Cis-acting DNA sequences, or DNA target elements, can be inserted into the MCS and used as baits to screen GAL4 AD/cDNA fusion libraries for proteins that interact with the target sequence. A protein-DNA (or one-hybrid) interaction can be detected by performing the assay in a yeast strain such as Y187 that is auxotrophic for histidine. Positive one-hybrid interactions drive expression of the HIS3 reporter gene, which enables the host cell to grow on histidine-deficient media.In the absence of activation, the constitutive HIS3 expression from PminHIS3 is very low. During library screening, the leaky expression of HIS3 is controlled by adding 3-amino-1,2,4-triazole (3-AT) to the medium. The concentration of 3-AT needed to fully suppress leaky HIS3 expression must be determined empirically for each DNA target element.pHIS2 can be maintained in both yeast and bacteria. It contains an autonomous replication sequence (ARS4) and TRP1 nutritional marker for replication and selection in yeast (1, 2); it contains a Col E1 origin and a kanamycin resistance gene (Kanr) for propagation and selection in E. coli. The centromeric sequence CEN6 ensures proper segregation of the plasmid during cell division in yeast (1, 2).To use pHIS2 in a one-hybrid assay, clone one or more copies of a cis-acting DNA target sequence into the MCS. Then introduce the plasmid into competent yeast cells using the transformation protocol in the BD Matchmaker Library Construction & Screening Kits User Manual (PT3529-1). In contrast to the original BD Matchmaker One- Hybrid System, this reporter vector does not need to be integrated into the yeast genome. Instead, it is maintained as an episome throughout the assay. Inserting your target element may alter the level of background HIS3 expression. Therefore, constructs should be tested for background (leaky) HIS3 expression before you start a one-hybrid analysis. Background growth due to leaky HIS3 expression is controlled by adding 3-AT to the selection medium, as described in the User Manual (PT3529-1).
| 载体基本信息 | |||
| 载体名称 | pHIS2 | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 7207 bp | 载体类型 | Yeast one hybrid systems |
| 克隆方法 | Enzyme digestion and ligation | ||
pHIS2 载体载体图谱
pHIS2 载体载体序列
LOCUS Exported 7207 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 7207)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..7207
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 152..814
/codon_start=1
/gene="S. cerevisiae HIS3"
/product="imidazoleglycerol-phosphate dehydratase, required
for histidine biosynthesis"
/note="HIS3"
/note="yeast auxotrophic marker"
/translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
promoter 2576..2857
/gene="S. cerevisiae TRP1"
/note="TRP1 promoter"
CDS 2858..3532
/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(3614..3632)
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(3653..3669)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3677..3693
/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(3701..3731)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 3746..3767
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(4055..4643)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4814..5608)
/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
GLAPAELFARLKASMPDGEDLVVTHDDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(5609..5713)
/gene="bla"
/note="AmpR promoter"
misc_feature 5746..6247
/note="CEN/ARS"
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
rep_origin complement(6564..7019)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 7164..7180
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 7187..7205
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
