Oxford Genetics/pSFMinCMVFrCFP (OG575) Minimal CMV CFP Cloning Plasmid/OG575/1 Ea

PlasmidInfo:

PlasmidPurpose:

TheFrostyCyanFluorescenceProtein(FrCFP)isunderregulatorycontroloftheminimalCMVpromoterinthisplasmidgivinglowlevelexpressionoftheFrCFPtransgeneinmosttypesofmammaliancells.Positiveornegativeregulatoryelementscanbeintroducedupstreamoftheminimalpromtoerusingaspecially-positionedMCStoendowyourrequiredresponsiveness.Forexampletranscriptionfactorbindingsitescouldbeintroducedtoprovidetissue-selectivetransgeneexpression.Similarlydrug-responsivesitescouldbeintroducedtoprovideexpressionregulatedbyexternally-applieddrugs.

ThisplasmidcontainstheminimalCMVimmediateearlypromoterwithamultiplecloningsiteimmediatelyupstreamthatallowstranscriptionfactorbindingsitestobeinserted.Thisallowstissuespecificorphysiologicallyresponsivepromoterstobecreated.

SequenceandMap:

OtherInfo:

Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.

Cloning:

Thisplasmidcontainsagenewithinthemainmultiplecloningsite(NotI-ClaI).Anyplasmidthatwesellwherethegeneisthisconfigurationwillbelocatedintheexactsamepositioninrelationtothestartandstopcodonofthegene.TheonlyexceptionstothisrulearefusionsproteinswherethefusiongenemaybepositionedatthefrontorendoftheMCStoallowgenefusion.

Bypositioningallofourgenesinthesamelocationitallowsthemtobetransferredbetweenplasmidsusingthesamecloningmethodandrestrictionsitesregardlessoftheplasmidbeingusedfromourproductrange.Insertinganewgeneintothisplasmidshouldbeeasilypossibleusingarangeofstandardrestrictionenzymesitesthatflankthegenecurrentlyinthevector.

InthemultiplecloningsitetherearetwoimportantrestrictionsitescalledBsgIandBseRIsites.ThesesitesbothcuttheDNAatthesamepositionandcleavethestopcodonofthegeneinthemultiplecloningsiteinthisplasmidtherebyproducingaTAoverhang.Thisoverhangiscompatiblewithanyofourpeptideorreporterfusiontagplasmidsalsocutwitheitheroftheseenzymes.ThisallowsseamlessC-terminalfusionstobemadewiththegeneinthismultiplecloningsiteusingasinglecloningstepfromourC-terminalpeptideandreportertagproductrange.NormallytheeasiestmethodistoclonetheC-terminaltagfromourotherplasmidproductsintothisplasmidusingBsgIorBseRIandthedownstreamClaIrestrictionsite.

BseRIandBsgIsitesarenon-palindromicandcleaveadefinednumberofbasesawayfromtheirbindingsites.Thisallowsthemtocuttheupstreamstopcodoninthegeneinthisplasmidregardlessofthegenesequence.

IPStatus:

ThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere