pYD1 is a 5.0 kb expression vector designed for expression, secretion, and display of proteins on the extracellular surface of Saccharomyces cerevisiae cells. The vector contains the following elements:1. AGA2 gene from Saccharomyces cerevisiae. This gene encodes one of the subunits of the a-agglutinin receptor. Fusion of the gene of interest to AGA2 allows secretion and display of the protein of interest. 2. GAL1 promoter for regulated expression of the AGA2 gene fusion. 3. Xpress epitope and V5 epitope for detection of the displayed protein. 4. Polyhistidine (6xHis) tag for detection and possible purification on metal chelating resin. 5. TRP1 gene for selection in Saccharomyces cerevisiae. 6. CEN6/ARS4 for stable, episomal replication in yeast. 7. Ampicillin resistance gene and the pUC origin for selection and replication in E. coli.
| 载体基本信息 | |||
| 载体名称 | pYD1 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 5009 bp | 载体类型 | Yeast Plasmids |
| 载体来源 | Invitrogen | 筛选标记 | TRP1 |
| 拷贝数 | High copy number | 启动子 | GAL1 promoter |
| 克隆方法 | Enzyme Cut | 5引物 | pYD1-F: AGTAACGTTTGTCAGTAATTGC |
| 3引物 | pYD1-R: GTCGATTTTGTTACATCTACAC | ||
pYD1 载体载体图谱
pYD1 载体载体序列
LOCUS Exported 5009 bp ds-DNA circular SYN 11-SEP-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pYD1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5009)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5009
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 2..443
/gene="S. cerevisiae GAL1"
/note="GAL1 promoter"
/note="inducible promoter, regulated by Gal4"
protein_bind 2..119
/bound_moiety="Gal4"
/note="UAS"
/note="upstream activating sequence mediating
Gal4-dependent induction"
promoter 475..493
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 858..887
/codon_start=1
/product="leader peptide from bacteriophage T7 gene 10"
/note="T7 tag (gene 10 leader)"
/note="promotes efficient translation in E. coli"
/translation="ASMTGGQQMG"
CDS 891..914
/codon_start=1
/product="Xpress(TM) epitope tag, including an enterokinase
recognition and cleavage site"
/note="Xpress(TM) tag"
/translation="DLYDDDDK"
CDS 993..1034
/codon_start=1
/product="epitope tag from simian virus 5"
/note="V5 tag"
/translation="GKPIPNPLLGLDST"
CDS 1044..1061
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
CDS complement(1433..2107)
/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(2108..2209)
/gene="S. cerevisiae TRP1"
/note="TRP1 promoter"
misc_feature 2286..2789
/note="CEN/ARS"
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
promoter 2826..2930
/gene="bla"
/note="AmpR promoter"
CDS 2931..3791
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 3962..4550
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 4874..4904
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 4912..4928
/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)."
primer_bind 4936..4952
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
promoter 4973..4991
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
