Basic Vector Information
The pTYB1 is an E. coli vector created for fusion protein expression and purification. A target gene can be inserted into the the ORF by use of the MCS upstream of the Sce VMA intein. The NdeI site in the MCS has the initiator sequence ATG that enables translation of the ORF. The chitin-binding domain(CBD) downstream of the intein results in a target protein-intein with C-terminal chitin beads that facilitate protein purification. Release of the protein is induced by the thiol cleavage activity of the intein.
pTYB1 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 ℃.
pTYB1 vector Sequence
LOCUS pTYB1. 7477 bp DNA circular SYN 18-SEP-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS pTYB1.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7477)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 7477)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7477
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 35..139
/label=AmpR promoter
CDS 140..997
/label=AmpR
/note="beta-lactamase"
rep_origin complement(1042..1555)
/direction=LEFT
/label=M13 ori
/note="M13 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
rep_origin 1666..2254
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(2626..2814)
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
protein_bind complement(3337..3358)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(3374..4453)
/label=lacI
/note="lac repressor"
promoter complement(4454..4531)
/label=lacI promoter
terminator 4684..4770
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 4867..4953
/gene="Escherichia coli rrnB"
/label=Escherichia coli rrnB terminator
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 5050..5136
/gene="Escherichia coli rrnB"
/label=Escherichia coli rrnB terminator
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 5233..5319
/gene="Escherichia coli rrnB"
/label=Escherichia coli rrnB terminator
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator complement(5419..5462)
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
promoter 5637..5655
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 5656..5680
/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)."
RBS 5695..5717
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 5776..6594
/label=Sce VMA intein 5' region
/note="5' region of the intein from the yeast Vma1 subunit
of the vacuolar ATPase (Chong et al., 1998)"
CDS 6598..7131
/label=Sce VMA intein 3' region
/note="modified 3' region of the intein from the yeast Vma1
subunit of the vacuolar ATPase (Chong et al., 1998)"
CDS 7171..7326
/label=CBD
/note="chitin binding domain from chitinase A1 (Watanabe et
al., 1994)"
terminator 7405..7452
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
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