Basic Vector Information
- Vector Name:
- pNMB2
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8011 bp
- Type:
- Yeast display vector
- Replication origin:
- ori
- Host:
- Yeast
- Source/Author:
- Uchanski T, Masiulis S, Fischer B, Kalichuk V
- Promoter:
- GAL1,10
pNMB2 vector Vector Map
Plasmid Resuspension 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 ℃.
pNMB2 vector Sequence
LOCUS 62056_18110 8011 bp DNA circular SYN 07-JAN-2021
DEFINITION Yeast display vector pNMB2, complete sequence.
ACCESSION MT338523
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8011)
AUTHORS Uchanski T, Masiulis S, Fischer B, Kalichuk V, Lopez-Sanchez U,
Zarkadas E, Weckener M, Sente A, Ward P, Wohlkoenig A, Zoegg T,
Remaut H, Naismith JH, Nury H, Vranken W, Aricescu AR, Pardon E,
Steyaert J.
TITLE Megabodies expand the nanobody toolkit for protein structure
determination by single-particle cryo-EM
JOURNAL Nat Methods 18 (1), 60-68 (2021)
REFERENCE 2 (bases 1 to 8011)
AUTHORS Steyaert J, Uchanski T, Pardon E.
TITLE Direct Submission
JOURNAL Submitted (14-APR-2020) VIB-VUB Center for Structural Biology, VIB,
Pleinlaan 2, Brussels 1050, Belgium
REFERENCE 3 (bases 1 to 8011)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Methods"; date: "2021"; volume: "18"; issue: "1"; pages: "60-68"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(14-APR-2020) VIB-VUB Center for Structural Biology, VIB, Pleinlaan
2, Brussels 1050, Belgium"
COMMENT ##Assembly-Data-START##
Sequencing Technology :: Sanger dideoxy sequencing
##Assembly-Data-END##
FEATURES Location/Qualifiers
source 1..8011
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature complement(70..573)
/label=CEN/ARS
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
promoter 610..714
/label=AmpR promoter
CDS 715..1572
/label=AmpR
/note="beta-lactamase"
rep_origin 1746..2334
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 2622..2643
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2658..2688
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2696..2712
/label=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 2720..2736
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 2757..2775
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 2940..3592
/label=GAL1,10 promoter
/note="divergent inducible promoter, regulated by Gal4"
misc_feature 3632..3898
/label=appS4 leader sequence
/note="appS4 leader sequence"
misc_feature 3899..3937
/label=nanobody beta-strain A (residues 1-14)
/note="nanobody beta-strain A (residues 1-14)"
misc_feature 3938..5161
/label=cHopQ scaffold protein
/note="cHopQ scaffold protein"
misc_feature 5162..5200
/label=multi cloning site
/note="multi cloning site"
misc_feature 5201..5302
/label=linker
/note="linker"
misc_feature 5303..5509
/label=Aga2p anchor protein
/note="Aga2p anchor protein"
CDS 5531..5764
/label=ACP
/note="acyl carrier protein (ACP) from E. coli"
misc_feature 5768..5797
/label=Myc-tag
/note="Myc-tag"
regulatory 5811..6117
/label=MFa1
/note="MFa1"
/regulatory_class="terminator"
promoter complement(6132..6150)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(6157..6173)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 6318..6773
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(6873..7544)
/label=TRP1
/note="phosphoribosylanthranilate isomerase, required for
tryptophan biosynthesis"
promoter complement(7545..7825)
/label=TRP1 promoter
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