pBAD18-Kan (ATCC® 87397)

Applications: expression vectorproduces protein arabinose regulatorvector containing primer sites useful for sequencing  /  Depositors: L Guzman, J Beckwith

Designations pBAD18-Kan
Depositors L Guzman, J Beckwith
Biosafety Level 1
Host
Distribution host: Escherichia coli DH5alpha
Vector Information
Size (kb): 5.4369997978210450
DESCRIPTION OF VECTOR:
Intact vector size: 5.437
Type of vector: phagemid
Cloning sites: NheI EcoRI SacI KpnI XbaI SalI PstI SphI
Polylinker sites: NheI EcoRI SacI KpnI SmaI BamHI XbaI SalI AccI PstI SphI
HindIII
Construction: pBAD18, kanR (pUC4K)
Host range: Escherichia coli
Features (with orientation and position when available):
regulator: araC, <-, 3346-4224
operator: O2, 4253-4270
promoter: araC, <-, 4375-4403
operator: O1, 4411-4432
other: CAP site, 4454-4467
operator: I2 + I1, 4463-4501
promoter for expression: arabinose BAD, ->, 4500-4527
MCS: NheI...HindIII, ->, 4550-4612
transcription terminator: rrnB T1 + T2, ->, 4613-5038
marker(s): kanR, ->, 123-938
replicon: M13, 1417-1875
replicon: pMB1, 1881-2578
Vector: pBAD18-Kan (phagemid)
Promoters: Promoter araC
Construction: pBAD18, kanR (pUC4K)
Marker(s):kanR,araC
Construct size (kb): 5.436999797821045
Features: marker(s): kanR
operator: I2 + I1
operator: O1
operator: O2
other: CAP site
promoter: araC
promoter for expression: arabinose BAD
regulator: araC
replicon: M13
replicon: pMB1
MCS: NheI...HindIII
transcription terminator: rrnB T1 + T2
Applications
expression vector
produces protein arabinose regulator
vector containing primer sites useful for sequencing
Comments
Restriction digests of the clone give the following sizes (kb): EcoRI--5.4; HindIII--3.9, 1.5; PstI--5.4.
Cultures should be grown in minimal media for more reproducible induction of expression. Expression is induced in glycerol-containing media by addition of arabinose. Expression is repressed by addition of glucose or other catabolites.
One of several tightly controlled expression vectors (ATCC 87393-87402) regulated by the arabinose operon. The vectors differ in replicon, antibiotic resistance marker, multiple cloning site and mechanism of initiation of translation.
The following primers can be used for sequencing of cloned inserts: 5' primer (27 - 8 bp upstream of the NheI site) 5'-CTGTTTCTCCATACCCGTT-3'; and one of two 3' primers: 3' primer 1 (2 - 19 bp downstream of the HindIII site) 5'-CTCATCCGCCAAAACAG-3';
3' primer 2 (17 - 33 bp downstream of the HindIII site) 5'-GGCTGAAAATCTTCTCT-3'.
Cloned inserts must provide a translation initiation sequence (ATG) and ribosome binding site for expression.
Plasmid contains bla (ampR) sequences surrounding the kanR gene which could promoter recombination if this plasmid is used in combination with other compatible ampR plasmids.
Recombination can be avoided by the use of recA host strains, or it can be used to advantage to intentionally exchange markers among plasmids.
Media ATCC® Medium 1236: LB Medium (ATCC medium 1065) with 25 mcg/ml kanamycin
Growth Conditions
Temperature: 37.0°C
References

Guzman LM, et al. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177: 4121-4130, 1995. PubMed: 7608087

Shipped frozen
Shipping Information Distributed: freeze-dried
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