Basic Vector Information
pSC101 ori is a low-copy replication origin that requires the Rep101 protein, which is a temperature-sensitive protein. When bacteria containing a plasmid with this ori is cultured at 37℃, the plasmid will be lost.
- Vector Name:
- pBAD43
- Antibiotic Resistance:
- Spectinomycin
- Length:
- 6179 bp
- Type:
- E.coli expression plasmid
- Replication origin:
- pSC101 ori
- Source/Author:
- Beckwith Lab
- Copy Number:
- Low copy number
- Promoter:
- araBAD
- Cloning Method:
- Enzyme Cut
- 5' Primer:
- pBAD-F: ATGCCATAGCATTTTTATCC
- 3' Primer:
- pBAD-R: gatttaatctgtatcagg
- Expression Method:
- L-arabinose Induced
pBAD43 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 ℃.
pBAD43 vector Sequence
LOCUS 40924_5869 6179 bp DNA circular SYN 22-SEP-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6179)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 6179)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6179
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(194..1069)
/codon_start=1
/label=araC
/note="L-arabinose regulatory protein"
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"
promoter 1096..1380
/label=araBAD promoter
/note="promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)"
terminator 1664..1750
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 1842..1869
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
primer_bind complement(1950..1966)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
CDS complement(2492..3280)
/codon_start=1
/label=SmR
/note="aminoglycoside adenylyltransferase (Murphy, 1985)"
/translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKEVAADWAMERLPAQYQP
VIREARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
CDS complement(4317..5264)
/codon_start=1
/label=Rep101
/note="RepA protein needed for replication with the pSC101
origin"
/translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
rep_origin complement(5312..5534)
/direction=LEFT
/label=pSC101 ori
/note="low-copy replication origin that requires the Rep101
protein"
protein_bind 6099..6120
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6135..6165
/label=lac promoter
/note="promoter for the E. coli lac operon"
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