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pSIT

87064

Clone type
Vector
Applications
Molecular biology
Product format
Freeze-dried
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Documentation

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

Detailed product information

General

Specific applications
expression vector
vector permitting RNA synthesis in vitro
vector permitting production of single-stranded DNA
vector useful for site-directed mutagenesis

Characteristics

Comments
Restriction digests of the clone give the following sizes (kb): BamHI--6.1; XbaI--6.1; EcoRV--4.35, 1.75.
Vector contains the following restriction sites (approximate kb from nt 1): BamHI--0.14; BglI--1.40, 1.63, 4.47, 4.59, 6.04; ClaI--0.49; EcoRV--0.65, 2.40; NcoI--0.11; PvuI--1.86, 4.72, 6.07; PvuII--1.89, 2.16, 2.25; XbaI--0.07.
Mutagenesis is achieved through alkali denaturation of the plasmid containing a cloned insert, followed by amplification of the plasmid using an ampicillin repair primer and a mutagenic primer.
Single stranded DNA can also be generated by infection with a helper phage such as R408 or M13K07 (ATCC 37468).
Following in vitro synthesis of the second DNA strand, the plasmid should be grown in E. coli BMH 71-18 mutS or ES1301 mutS (to avoid repair of the mutation) and can be selected for ampicillin resistance.
Clones can then be transformed into a suitable strain for propagation (E. coli JM109 - ATCC 53323) or expression (E. coli BL21 (DE3)).
Several rounds of mutation can be achieved by including a tetracycline inactivation primer in the first round, so that tetracycline resistance can be restored and used as a marker for second round mutants.
Best mutagenic efficiency was achieved using a 10 fold molar excess of mutagenic primer to ampicillin repair primer.
Expression vector allowing site directed mutagenesis of cloned inserts and subsequent selection of mutants for ampicillin resistance.
The following oligonucleotides can be used to repair or inactivate the vector markers: ampicillin repair, 5'-GTTGCCATTGCTGCAGGCATCGTGGTG-3'; ampicillin knockout, 5'-GTTGCCATTGCGGCATCGTGGTGTCAC-3'; tetracyclin repair, 5'-GCCGGGCCTCTTGCGGGATATCGTCCA-3';
tetracycline knockout, 5'-GCCGGGCCTCTTGCGGGCGTCCATTCC-3'.
Constructed from pSELECT by replacing the lacZ region with a T7 promoter, under control of the lac repressor, and replacing the rop copy number control sequence with the lacIq repressor gene.
Mycoplasma contamination
Not detected

Vector information

Construct size (kb)
6.099999904632568
Vector name
pSIT (phagemid)
Construction
pSELECT, T7 promoter (pET-11d)
initiation codon
ATG
Markers
tetR
Operator
lac
Promoters
T7 (phi10)
Replicon
f1; pMB1
Repressor gene
lacIq
Restriction sites
BamHI; EspI; NcoI
Ribosome binding site
T7 (phi10)
Terminator
T7 (phi10)

History

Depositors
E Hunter
Special collection
NCRR Contract

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

Frequently Asked Questions

References

Curated Citations

Andreansky M, Hunter E. Phagemid pSIT permits efficient in vitro mutagenesis and tightly controlled expression in E. coli. BioTechniques 16: 626-633, 1994. PubMed: 8024782

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