PlasmidInfo:
PlasmidInformation
ProductName:pSF-Lenti
ProductCode:OG269
Size(bp):7832bp
BacterialAntibioticSelection:
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Cytomegalovirus(CMV)immediateearlypromoter/MousePhosphoglycerateKinase(PGK)Promoter
PlasmidPurpose:
ThisplasmidcontainsaHIV1basedlentivirusexpressionvectorthatcanbeusedforthestableintegrationofgenesintobothdividingandnon-dividingcells.TheplasmidisbasedonthecommonlyusedpCCLlentivirusbackboneandcontainsarangeofmodificationstomakecloningeasier.ThevectorcontainsanexpandedmultiplecloningsiteandiscompatIBLewithmostoftheplasmidsweprovideinourproductrangewhichallowssectionstocompiledtocreatecomplexexpressionvectors.ThisplasmidcontainstheCMVpromotertodrivetheexpressionofageneofinterestfollowedbyamultiplecloningsiteandthePGKpromotertodrivepuromycinresistance.
PromoterExpressionLevel:ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.Forthisreasonwestockarangeofotherpromotersthatarecompatiblewiththisplasmidandareavailableonrequest.Themurinephosphoglyceratekinase(PGK)promoterdrivingpuromycinexpressionallowsforlongtermstableexpressioninvitroandinvivoinmosttypes.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Transcriptionisterminatedviaapolyadenylationsignalwithinthe3primeLTRofthelentivirusgenome.
Cloning:
CloninginaGene:Thisplasmidhasbeendesignedtobecompatiblewitharangeofcloningtechniques.Themultiplecloningsitecontainsarangeofstandardcommonlyusedrestrictionsitesforcloning.UsingthesesitesgenescanbeinsertedusingstandardcloningmethodswithDNAligase.Othermethodssuchasligaseindependentcloning(LIC)GibsonAssemblyInFusionHDorSeamlessGeneArtcanalsobeusedandbecauseallofourplasmidsarebasedonthesamebackbonethesamemethodcanbeusedforcloningintoallofourcataloguevectors.
Multiplecloningsitenotes:ThereareafewimportantsiteswithintheMCS.TheseincludetheNcoIsitetheXbaIsiteandtheBsgIandBseRIsites.TheNcoIsitecontainsastartcodonthatisimmediatelydownstreamofbothaKozakandShine-Dalgarnoribosomalbindingsite.Theseallowforoptimalpositioningofgeneswhenthestartcodonisplacedinthislocation.IfthisisnotrequiredandyouwishtouseadownstreamsiteforgenecloningyoucanremovetheNcoIsitebycleavingtheplasmidwithKpnI.
TheXbaIsitecontainsastopcodon.ThisstopcodonispositionedinaspecificpositioninrelationtotheBsgIandBseRIsitesthatareimmediatelydownstream.WheneitherBseRIorBsgIcleavetheplasmidtheyproduceaTAoverhangfromthestopcodonintheXbaIsitethatiscompatiblewithallofourpeptidetagplasmidscutwiththesamesites.BseRIandBsgIsitesarenon-palindromicandcleaveadefinednumberofbasesawayfromtheirbindingsite.
WheneverwecloneageneintoourmultiplecloningsitewealwayspositionthestartandstopcodoninthesamepositionsintheMCS.IfthestartandendsofthegenesarenotcompatiblewithNcoIandXbaIweextendthesequencetothenearestexternalsitesbutkeepthestartandstopcodonslocationsconsistent.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastProplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
