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当前位置: 首页 > 产品中心 > Sandwich_method_ELISA > Oxford Genetics/pSF-T7-LacO-COOH-3C-Rluc-6His (OG4470)/OG4470/1 Ea
商品详细Oxford Genetics/pSF-T7-LacO-COOH-3C-Rluc-6His (OG4470)/OG4470/1 Ea
Oxford Genetics/pSF-T7-LacO-COOH-3C-Rluc-6His (OG4470)/OG4470/1 Ea
Oxford Genetics/pSF-T7-LacO-COOH-3C-Rluc-6His (OG4470)/OG4470/1 Ea
商品编号: OG4470
品牌: oxgene
市场价: ¥5321.60
美元价: 3192.96
产地: 美国(厂家直采)
公司:
产品分类: 夹心法ELISA
公司分类: Sandwich_method_ELISA
联系Q Q: 3392242852
电话号码: 4000-520-616
电子邮箱: info@ebiomall.com
商品介绍

PlasmidInfo:

PlasmidInformation

ProductName:pSF-T7-LacO-COOH-3C-Rluc-6His

ProductCode:OG4470

Size(bp):6374bp

BacterialAntibioticSelection:KanR

OriginandCompatibility:pUChighcopyderivedfrompBR322

BacterialCopyNumber:500-700percell

Promoter:

PlasmidPurpose:

ThisplasmidisdesignedtoexpresstaggedproteinsinE.coli.ItcontainstheT7promoterthatcanberegulatedusingtheLacrepressor.ThisallowsforgreatercontroloverexpressionincomparisontonormalT7promoterplasmids.InordertousethisplasmidyouwillrequireaT7polymeraseexpressingE.colicelllinethatalsoexpressestheLacIrepressor.AcommonexpressioncelllinethatcanbeusedforthispurposeistheBL21(DE3)strain.

AbouttheCleavageTag:

Thisplasmidalsoencodesaproteasecleavagesitethatisdesignedtobepositionedbetweenyourgeneofinterestandthetagtoallowtheremovalofthetagfollowingproteinpurificationorisolation.Thisplasmidcontainsa3Ccleavagetag.Theproteinsequenceofthecleavagetagis:LEVLFQ?GP.HumanRhinovirus(HRV)3CProteaseisahighlyspecificproteasethatcleavesbetweentheGluandGlyresiduesofitsrecognitionsite.Itisoftenproducedwiththetrademname"PreScissionprotease".

Formoreinformationonwhichcleavagetagtouseseeourcleavagetagguide.

PromoterExpressionLevel:

ThisplasmidcontainstheIPTGinducIBLepromoterthatwascreatedbythefusionoftheLacpromoterandtheTryptophanoperonpromoter.ItallowsinducibleexpressioninE.coliusingIPTGastheinducingagent.

AboutthePeptideTag:

Thisplasmidcontainsac-terminalRenillaluciferasereportertagthatcanbefusedtoageneofinteresttoallowproteindetection.

ThisplasmidalsocontainsasecondaryHexa-Histidine(6His)tagproteintag.Thesequenceofthistagis:HHHHHH

Weprovidearangeofdualpeptidetagplasmids.ThisisbecausesomepeptidetagsprovidespecificBIOLOGicalproperties(e.gsmallmoleculeaffinitynewepitopessolubilityorproteinsecretion)thatarenotprovidedbyothers.

SequenceandMap:

OtherInfo:

TranscriptionTermination:

ThisplasmidcontainsthreealternativetranscriptionterminatorsforTagT7LacOInduciblebacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.

Cloning:

MakingProteinFusions:

ThisplasmidhasbeendesignedtoallowthreetypesofcloningintothemainMCStojoinacodingsequencewiththetag.

CloninginaGene:

Thisplasmidhasbeendesignedtobecompatiblewitharangeofcloningtechniques.Themultiplecloningsitecontainsarangeofstandardcommonlyusedrestrictionsitesforcloning.UsingthesesitesgenescanbeinsertedusingstandardcloningmethodswithDNAligase.Othermethodssuchasligaseindependentcloning(LIC)GibsonAssemblyInFusionHDorSeamlessGeneArtcanalsobeusedandbecauseallofourplasmidsarebasedonthesamebackbonethesamemethodcanbeusedforcloningintoallofourcataloguevectors.

Multiplecloningsitenotes:

ThereareafewimportantsiteswithintheMCS.TheseincludetheNcoIsitetheXbaIsiteandtheBsgIandBseRIsites.TheNcoIsitecontainsastartcodonthatisimmediatelydownstreamofbothaKozakandShine-Dalgarnoribosomalbindingsite.Theseallowforoptimalpositioningofgeneswhenthestartcodonisplacedinthislocation.IfthisisnotrequiredandyouwishtouseadownstreamsiteforgenecloningyoucanremovetheNcoIsitebycleavingtheplasmidwithKpnI.

TheXbaIsitecontainsastopcodon.ThisstopcodonispositionedinaspecificpositioninrelationtotheBsgIandBseRIsitesthatareimmediatelydownstream.WheneitherBseRIorBsgIcleavetheplasmidtheyproduceaTAoverhangfromthestopcodonintheXbaIsitethatiscompatiblewithallofourpeptidetagplasmidscutwiththesamesites.BseRIandBsgIsitesarenon-palindromicandcleaveadefinednumberofbasesawayfromtheirbindingsite.

WheneverwecloneageneintoourmultiplecloningsitewealwayspositionthestartandstopcodoninthesamepositionsintheMCS.IfthestartandendsofthegenesarenotcompatiblewithNcoIandXbaIweextendthesequencetothenearestexternalsitesbutkeepthestartandstopcodonslocationsconsistent.

1:SnapFusionCloning:

IfyouwouldliketofuseyourcodingsequencetothetagwithminimaladditionalbasesyoucanuseourSnapFusiontechnology.ThisprocessinvolvesamplifyingyourgenebyPCRtoaddspecificrestrictionsitesontotheends.WhenthesesitesarecuttheyproduceanoverhangthatiscompatiblewiththisplasmidcutwithBseRIorBsgI.

Toinsertyourgene:


1:Amplifyyourgenewithprimersdesignedusingthisspreadsheet
2:CuttheplasmidwitheitherBseRIorBsgI.*
3:Cutyourgenewiththeenzymeyouaddedusingthespreadsheet(anyofAcuIBpmIBpuEIBseRIBsgIEciI).
4:ClonethegeneintotheplasmidusingDNAligase.

UsingthismethodwithanN-terminaltagplasmidwillresultinthetagcodingsequenceimmediatelyfollowedbyyourgenesATGstartcodonatthejoin.Thisresultsinaseamlessfusionofthetwosequenceswithnoextrabasesbeingadded.UsingthismethodonC-terminaltagplasmidswillconvertyourgenesstopcodonintoaTAC(TyrY)codonfollowedbytheplasmidtagcodingsequence.Thisresultsinnoextrabasesbetweenyourgeneandthetag.Seethediagrambelowformoreinformation.

*PleasenotethatinsectexpressionplasmidscannotbecutwithBsgIonlyBseRIbecauseofunavoidableconflictingsitesinthebackbone.AlsoYeastplasmidscannotbecutwithBseRIbecauseofunavoidablerestrictionsitesinthebackbone.

Usingthistechniquewillcreateagenefragmentthatcanbeligatedintoanyorour>1500peptideandreportertagplasmids.Ifyouuseoneoftheothertechniquesbelow(GibsonInFusionSeamlessorLIC)youwillneednewprimersforeveryvectoryoucloneintobecausethearmsofhomologywillchangeaccordingtothetagplasmidyouarecloninginto.

Ifyoufindthatyourgenesequencehassitesinitthatmakeusingthiscloningstrategydifficultyoucanstilluseoneofthealternativemethodsbelow(e.g.standardcloningorGibsoncloning).

OpenthePrimerDesignTooltohelpyoudesignprimersforcloningyourgeneinourSnapFusiontechnique.

2:StandardEnzymes:

Ifyouarenotconcernedaboutleavingafewextrabasesbetweenthetagcodingsequenceandyourgeneyoucancloneyourgeneintothevectorusingstandardcloningrestrictionenzymes.Thisstrategywillrequireyoutochoosewhichenzymesyouwanttousetocloneyourgene.

OpenthePrimerDesignToolwhichprovidesprimerswithdifferentenzymechoicespositioningyourgeneasclosetothetagaspossibleineachcase.Pleasenotethatstandardenzymeswillalwaysleaveadditionalnucleotidesbetweenyourgeneandthetagbutusingthespreadsheetwillensurethetagandgeneareinframe.

3:Gibsoncloning/InfusionHD/GeneArtSeamless/LigaseIndependentCloning(LIC)Methods:

ThesecloningtechniquesusereagentssoldbyothercompaniesandallowyoutofusesequencestogetherusingenzymesthatchewbacktheDNAtoleaveoverlappingends/overhangs.ThesubsequentmethodofjoiningtheDNAdependsonthekitused.Touseoneofthesetechniquesyoucaneitherdesignyourownprimersoryoucanusethespreadsheetbelowtohelpwiththedesign.

OpenthePrimerDesignTooltohelpyoudesignprimersforcloningyourgeneusingGibsonassemblyInfusionHDGeneArtSeamlesscloningorLigaseIndependentCloning(LIC)techniques.

IPStatus:

IntellectualPropertyStatus

ThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere

品牌介绍
OXGENE的技术平台和专家解决方案加速了细胞和基因疗法的发现,开发和制造。我们解决了现代生物学中基因治疗,抗体治疗和基因编辑方面最重要和最具挑战性的问题。我们的技术可以实现精确而强大的哺乳动物细胞工程。我们以自动化和信息学为导向的方法意味着我们解决了其他人无法推进新疗法交付的问题。