pDEST32 vector (V001687)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V001687	pDEST32	In stock, instant shipping	Buy one, get one free!

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

pDEST32 is used for the Yeast two hybrid system.

Vector Name:: pDEST32

Antibiotic Resistance:: Gentamicin

Length:: 12266 bp

Type:: Yeast Two-Hybrid System Vectors

Replication origin:: ori

Host:: Yeast

Promoter:: ADH1(long)

Growth Strain(s):: DB3.1

Growth Temperature:: 37℃

pDEST32 vector Map

Copy Sequence View Map

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5. Store the plasmid at -20 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

References

Rajagopala SV, Hughes KT, Uetz P. Benchmarking yeast two-hybrid systems using the interactions of bacterial motility proteins. Proteomics. 2009 Dec;9(23):5296-302.

Reece-Hoyes JS, Walhout AJM. Generating Yeast Two-Hybrid Bait Strains. Cold Spring Harb Protoc. 2018 Jul 2;2018(7).

pDEST32 vector Sequence

LOCUS       Exported               12266 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 12266)
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..12266
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        849..1553
                     /gene="S. cerevisiae ADH1"
                     /note="ADH1 promoter"
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             1581..2021
                     /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"
     protein_bind    2037..2161
                     /gene="mutant version of attR"
                     /bound_moiety="LR Clonase(TM)"
                     /note="attR1"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2308..2410
                     /note="cat promoter"
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             2411..3091
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /note="CmR"
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     CDS             3411..3716
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /note="ccdB"
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3757..3881)
                     /gene="mutant version of attR"
                     /bound_moiety="LR Clonase(TM)"
                     /note="attR2"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      4081..4268
                     /gene="S. cerevisiae ADH1"
                     /note="ADH1 terminator"
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     promoter        complement(4421..4439)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4446..4462)
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      4603..5058
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5359..5766
                     /gene="S. cerevisiae LEU2"
                     /note="LEU2 promoter"
     CDS             5767..6861
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /note="LEU2"
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     misc_feature    7594..8097
                     /note="CEN/ARS"
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(8452..8985)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /note="GmR"
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(9174..9202)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /note="Pc promoter"
                     /note="class 1 integron promoter"
     rep_origin      9859..10447
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    10735..10756
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding site"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        10771..10801
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    10809..10825
                     /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     10833..10849
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        10870..10888
                     /note="T3 promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
     terminator      10918..11165
                     /gene="S. cerevisiae CYC1"
                     /note="CYC1 terminator"
                     /note="transcription terminator for CYC1"