pTYB2 vector (V012776)

Basic Vector Information

pTYB2 is an E. coli cloning and expression vector (7474 bp) used in the IMPACT™ Kit (NEB #E6901) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag (1,2). This C-terminal fusion vector is designed for the in-frame insertion of a target gene into the polylinker upstream of an intein tag (the Sce VMA intein/chitin binding domain, 55 kDa) (1,2). This results in the fusion of the C-terminus of the target protein to the N-terminus of the intein tag. Thiol-induced self-cleavage of the intein releases the target protein from the chitin-bound intein tag, resulting in a single column purification of the target protein. For fusion of the N-terminus of the target protein to the intein tag, use pTYB21 (NEB #N6709) or pTYB22 (NEB #N6710). This vector can be used in conjuction with pTYB22 (NEB #N6710) to test which fusion constructin (N-terminal or C-terminal) maximizes the expression and yield of a target protein (3).

Vector Name:: pTYB2

Antibiotic Resistance:: Ampicillin

Length:: 7474 bp

Type:: E.coli expression plasmid

Replication origin:: ori

Copy Number:: High copy number

Promoter:: T7

Cloning Method:: Enzyme Cut

pTYB2 vector Map

Copy Sequence View Map

pTYB2 vector Sequence

LOCUS       pTYB2.        7474 bp DNA     circular SYN 18-SEP-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    pTYB2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7474)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7474)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7474
                     /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             5773..6591
                     /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             6595..7128
                     /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             7168..7323
                     /label=CBD
                     /note="chitin binding domain from chitinase A1 (Watanabe et
                     al., 1994)"
     terminator      7402..7449
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"

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