GenereplacementusingpKOvectors

DescriptionofpKO3

ExcerptsfromAndyLinks1994HarvardUniversityThesis,"ExperimentalToolsfortheAnalysisofGenomes"

ThevectorpKO3integratesintothechromosomebyhomologousrecombinationcreatingatandemduplicationatthenonpermissivetemperature.Whenshiftedtothepermissivetemperature,thepSC101replicationorigininthehostchromosomeisdetrimental,andthevectorisexcisedfromthechromosome.Toselectforthelossofvectorsequencefromthecell,theB.subtilisgenesacBwasincorporatedintothevector,sinceexpressionofsacBinthepresenceofsucroseisdetrimentaltoE.coli.

Becausetheentireprotocolcanbedonebyreplicaplatingontodifferentselectablemediumsatdifferenttemperatures,largenumbersofgenescanbesimultaneouslyreplacedwithknockoutalleles.UnlikeothermethodsusedforgenereplacementsinE.coliusingColE1plasmidsinapolA1background(GuttersonandKoshland,1983)ortransformationoflinearDNAintorecBCsbcBorrecDstrains(JasinandSchimmel,1984;Winansetal.,1985;Shevelletal.,1988),thisprotocolisperformeddirectlyinwild-typestrains.Moreover,sincethesystemisplasmidbased,genereplacementsareeasilyperformedinanygeneticbackgroundthatisrecombinationproficient.

TwoapproachesweredevelopedforgeneratingmutantallelesinvitrowhichcouldbeinsertedintothepKO3vectorforreplacingopenreADIngframesontheE.colichromosome.Bothmethodsweredevelopedwiththegoalofeventuallyapplyingthemtothelargenumberofunidentifiedopenreadingframes.ThefirstsetoftoolsinsertsanantibioticMarkerintorandomlyclonedsequencestodisrupttheopenreadingframe.Fromthisfirstmethod,asecondapproachwasderivedwhichusescrossoverPCRtogenerateprecisedeletionsoftheopenreadingframes.ThesetwomethodswereappliedtotwoE.colireadingframesidentifiedintheproteincontentsurveyofE.coli.

UsingpKO3

AndrewJ.Link,DerethR.Phillips,andGeorgeM.Church

HarvardMedicalSchool

DepartmentofGenetics

77AvenueLouisPasteur

Boston,MA02115

ThefollowingmethodsweredevelopedforworkingwiththegenereplacementvectorpKO3.TheplasmidandgenereplacementsprotocolswerederivedfromHamiltonetal.(1989)(J.Bacteriology171:4617-4622).

Atthenonpermissivetemperature,alteredchromosomalsequencescarriedonthepSC101plasmidintegratesintothechromosomebyhomologousrecombinationtocreateanimperfecttandemduplication.Whenthecellsareshiftedtothepermissivetemperature,thecointegranttendstoundergoesasecondrecombinationeventregeneratingtheplasmidinthecell.Dependingonthesiteofthesecondrecombination,eitherthewild-typeorthemutantalleleisleftbehindinthechromosome.Toselectforthelossofvectorsequencefromthecell,theB.subtilisgenesacBwasincorporatedintothevector,sinceexpressionofsacBinthepresenceofsucroseisdetrimentaltoE.coli.

Strain: ThegenereplacementexperimentsusedtherecombinationproficientstrainsEMG2(FlamBDa+)orMC1061(F-araD139del(araleu)7696del(lacY74)galUgalKhsdrhsdM+strA).

Mediaandgrowthconditions: AllstrainsweregrowninLBmedium(1%(w/v)bactotryptone,0.5%(w/v)yeastextract,0.5%(w/v)NaCl)withtheappropriateselection.Forantibioticselection,theconcentrationofchromamphenicolwas20mg/ml.ForselectionagainstsacB,LBmediumwassupplementedwithsucrosetoafinalsucroseconcentrationof5%(w/v)

NOTE:pKO3hasatemperaturesensitivepSC101replicationorigin.Torecovertheplasmid,strainsharboringtheplasmidmustbegrownat30degCunderchloramphenicolselection.

IsolationofpKO3: E.colistrainsharboringpKO3plasmidaregrowninLBmediaat30degCunderchloramphenicolselectiontostationaryphase.PlasmidDNAisisolatedusuallythealkaline-lysismethod(BirnboimandDoly)orQiagensplasmidprepkit.

ForananalyticalrestrictiondigestofpKO3,1.5mlofovernightcultureisusedinthemini-prep,andtherecoveredplasmidDNAisresUSPendedin25ulofTE.5uloftheplasmidDNAisusedfortherestrictiondigestionanddetectiononethidiumbromidestainedagarosegels.ForpreparingpreparativeamountsofpKO3,50to1000mlofovernightcultureareused.ThepSC101plasmidispresentat5-10copiespercell.

Electroporation: 40mlofelectroporationcompetentcells(1x10^11cells/ml)weremixedwith1-3mlofDNAinaice-cold500ulmicrofugetubeandtransferredtoa0.2cmelectroporationcuvette(Biorad,Inc).Thecellsareelectroporatedat2,500kVwith25microfaradand200ohmresistance.Immediatelyafterelectroporation,1mlofSOC(2%bactotryptone,0.5%yeastextract,10mMNaCl,2.5mMKCl,10mMMgCl2,10mMMgSO4,20mMglucose)mediaisaddedtothecuvette.Thecellaretransferredtoa17x100mmpolypropylenetubeandallowedtorecoverfor1hat30degCshakenat250rpmbeforeplatingonselectivemedia.

DNAsequencing: Sequencingtheleftandrightvector-insertjunctionsofinsertsclonedintotheBamHIsiteofpKO3usedthesequencingprimerspK03-LandpK03-R.Cyclesequencingwasperformedessentiallyasdescribed(Murray,1989)usingtheStratagene"CyclistSequencing"kit(Stratagene,Inc.)andplasmidDNAisolatedusingaQiagenplasmidprepkit.Sequencingproductswerelabeledwithalpha-32P-dATP.

pK03-L:5-AGGGCAGGGTCGTTAAATAGC-3

pK03-R:5-TTAATGCGCCGCTACAGGGCG-3.

Polymerasechainreaction: PrimerspK03-LandpK03-RwereusedforPCRamplifyinginsertsclonedintotheBamHIsiteofpKO3.ThePCRreactionsusedeitherpurifiedDNAorbacterialcoloniesasthestartingtemplate.AllPCRreactionswereperformedinaPerkin-Elmer9600thermalcycler.PCRreactionbuffer(PonceandMicol(1992),NAR20:623)consistedof30mMtricine(pH8.4),2mMMgCl2,5mMbeta-mercaptoethanol,0.01%w/vgelatin,0.1%w/vThesit,200uMeachdNTP,600nMofeachprimer,and1unitTaqpolymerase(BoehringerMannheim).ThePCRreactionmixturewasdenaturedat94degCfor3minbeforeaddingtheTaqpolymerase.Thethermalcycleprofilewas15secat94degC,15secat55degC,and30secat72degC.Allexperimentsused30cycles,andafinal5min72degCholdstep.

Genereplacement: MutantallelesclonedintothepKO3genereplacementvectorareelectroporatedintorecombinationproficientstrains(eg.EMG2)andallowedtorecoverfor1hat30degC.Thecellsareplatedonprewarmedchloramphenicol/LBplatesandincubatedat42degC.Tomeasuretheintegrationfrequency,theelectroporatedcellsarealsoplatedonchloramphenicol/LBplatesat30degC.Fromthe42degCplate,1-5coloniesarepickedinto1mlofLBbroth,seriallydiluted,andimmediatelyplatedat30&degree;Coneither5%w/vsucroseor5%sucrose+antibioticplates.The5%sucroseplatesarereplicaplatedtochloramphenicolplatesat30degCtotestforlossofthereplacementvector(cms).ThegenereplacementisconfirmedbyeitherPCRusingprimersflankingthetargetedopenreadingframeorbygenomicSoutherns. 

IMPORTANTTHINGSTOREMEMBER:

1)  pK03hasatemperaturesensitivePsc101replicationorgin. Strainsharboringtheplasmidmustbegrownat30degCunderchloramphenicolselection.

2)  SacBappearstobemildlystressfultothecellevenwithoutaddedsucrose. ItisagoodideanottocarrytheplasmidfortoomanygenerationsinasinglestrainasyouwillaccumulatemutationseitherinthesacBgeneorinthebacterialgenometobypassthistoxicity.

3)  Makesureyoutestyourplasmidforallofthemarkers(ts,sacB,cat)beforeyoucloneanythingintoit. WegenerallycheckeachbatchofpK03beforeshipping. IntheabsenceofselectionthetsandsacBmarkersarebasicallystable. ThepK03strainhas"very-slow-growth"(NOT"no-growth")at43degoronsucrose,sostreaktestsarenotrecommended. Instead,wetakeapK03"test-colony"andseriallydiluteandplateforsinglecoloniesatboth30and40degrees. Ifthe43degreecoloniesaresmallerthanthe30degreecoloniesthenthe"test-colony"is"ts". Whenperformingtheknock-outprotocol,weonlypickthelargecoloniesonthe43degplates. Wedothesameserialdilutionwithsucrose/LBversusLBplatestotestforSacB.

4)  WearenotpreparedtoadviseresearchersontheuseofthisplasmidinanyotherspeciesorusinganycloningsitesotherthanthoseusedintheJ.Bact.Paper. Allotherusesareatyouwonrisk.

5)  ThepSC101plasmidisconsideredtobelowcopyandispresentat5-10copiespercell.Youmayneedtoadjustyourplasmidprepprotocolstoincreaseyouryield.

Foradditionalupdates,vectorandtagsequencesandinformationaboutE.colicommunitygeneknockoutresourcesharingpleaseconsultourwebsite: http://arep.med.harvard.edu/gmc/ecoko.html

pKO3&pKOVMapsandSequence

Figure1

 AmapofpKO3showingtheuniquerestrictionsites.Thesequencesusedtoconstructthemaphavenotbeenverified.WiththeexceptionofBamHI,NotI,andHindIII,therestrictionsiteshavenotbeenconfirmed.

Figure2

AdetailedmapoftheBamHIcloningsiteusedforcloninggenomicinsertsforgenereplacementexperimentsintheChurchlab.pK03-LandpK03-RareprimersequencesusedforPCRamplificationacrossthecloningsiteandcyclesequencingthevector-insertjunctions.

Sequencefiles(GCGformat):

GenereplacementusingthepKO3vector

 NOTE: The file is constructed from various GenBank files and is notprimary sequence and has not been confirmed.Comments:Sequence file of the gene replacement vector pKO3. The vector has a temperature-sensitive pSC101 replicon whichwhich is functional at 30 C.Hamiliton et. al. ,1989 J. Bacteriology 171: 4617-4622Link, A.J., Phillips, D. and Church, G.M. (1997) Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: Application to open reading frame characterization. J. Bacteriology 179: 6228-6237. Current base coordinates:221-880Cm-R; chloramphenicol acetyltransferase1119-1575f1 (+) filamentous origin2129-3550Bacillus subtilis sacB gene4139-4149oriC homologous region4204-4215IHF binding site4407-5357RepA (temperature-sensitive ?) Pko3.MailLength: 5681May 31, 199511:25Type: NCheck: 5438.. 1CCCTTTCGTC TTCGAATAAA TACCTGTGAC GGAAGATCAC TTCGCAGAAT 51AAATAAATCCTGGTGTCCCTGTTGATACCGGGAAGCCCTGGGCCAACTTT101TGGCGAAAATGAGACGTTGATCGGCACGTAAGAGGTTCCAACTTTCACCA151TAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTT201CAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACC251GTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTC301AGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCT351TTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATT401CACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAAT451GAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCG501TTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCAC551GACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTA601CGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTT651TCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTG701GCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTA751TACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATG801CCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAG851TACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTG901GTGCCCTTAAACGCCTGGTGCTACGCCTGAATAAGTGATAATAAGCGGAT951GAATGGCAGAAATTCGAAAGCAAATTCGACCCGGTCGTCGGTTCAGGGCA1001GGGTCGTTAAATAGCCGCTTATGTCTATTGCTGGTTTANTCGGTACCCGG1051GgatcgcggccgcggaccggatcctctagagcggccgcGATCCTCTAGAG1101TCGACCGGNGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCG1151CGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCC1201CTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGC1251CGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGAT1301TTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGT1351TCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTT1401GGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACAC1451TCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATT1501TCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAA1551TTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAA1601TGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGT1651ATCCGCTCATNNCANGATCCTTTTTAACCCATCACATATACCTGCCGTTC1701ACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTA1751AAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAA1801TGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAA1851TCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAA1901TCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTT1951TGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTA2001TACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTA2051ACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAAC2101ACAGTACATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAA2151AACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCA2201ACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATA2251CGGCATTTCCCATATTACACGCCATGATATGCTGCAAATCCCTGAACAGC2301AAAAAAATGAAAAATATCAAGTTCCTGAATTCGATTCGTCCACAATTAAA2351AATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACA2401AAACGCTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTG2451CATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTC2501TATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCG2551CGTCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAG2601ACCAAACACAAGAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAA2651ATCCGTTTATTCTACACTGATTTCTCCGGTAAACATTACGGCAAACAAAC2701ACTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAACA2751TCAACGGTGTAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAAACG2801TATCAAAATGTACAGCAGTTCATCGATGAAGGCAACTACAGCTCAGGCGA2851CAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGGCCACAAAT2901ACTTAGTATTTGAAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAA2951GAATCTTTATTTAACAAAGCATACTATGGCAAAAGCACATCATTCTTCCG3001TCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAAACGCACGGCTGAGT3051TAGCAAACGGCGCTCTCGGTATGATTGAGCTAAACGATGATTACACACTG3101AAAAAAGTGATGAAACCGCTGATTGCATCTAACACAGTAACAGATGAAAT3151TGAACGCGCGAACGTCTTTAAAATGAACGGCAAATGGTACCTGTTCACTG3201ACTCCCGCGGATCAAAAATGACGATTGACGGCATTACGTCTAACGATATT3251TACATGCTTGGTTATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGCT3301GAACAAAACTGGCCTTGTGTTAAAAATGGATCTTGATCCTAACGATGTAA3351CCTTTACTTACTCACACTTCGCTGTACCTCAAGCGAAAGGAAACAATGTC3401GTGATTACAAGCTATATGACAAACAGAGGATTCTACGCAGACAAACAATC3451AACGTTTGCGCCAAGCTTCCTGCTGAACATCAAAGGCAAGAAAACATCTG3501TTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGTTAACAAATAA3551AAACGCAAAAGAAAATGCCGATNNCCGGTTTATTGACTACCGGAAGCAGT3601GTGACCGTGTGCTTCTCAAATGCCTCAGGCTGTCTATGTGTGACTGTTGA3651GCTGTAACAAGTTGTCTCAGGTGTTCAATTTCATGTTCTAGTTGCTTTGT3701TTTACTGGTTTCACCTGTTCTATTAGGTGTTACATGCTGTTCATCTGTTA3751CATTGTCGATCTGTTCATGGTGAACAGCTTTAAATGCACCAAAAACTCGT3801AAAAGCTCTGATGTATCTATCTTTTTTACACCGTTTTCATCTGTGCATAT3851GGACAGTTTTCCCTTTGATATCTAACGGTGAACAGTTGTTCTACTTTTGT3901TTGTTAGTCTTGATGCTTCACTGATAGATACAAGAGCCATAAGAACCTCA3951GATCCTTCCGTATTTAGCCAGTATGTTCTCTAGTGTGGTTCGTTGTTTTT4001GCGTGAGCCATGAGAACGAACCATTGAGATCATGCTTACTTTGCATGTCA4051CTCAAAAATTTTGCCTCAAAACTGGTGAGCTGAATTTTTGCAGTTAAAGC4101ATCGTGTAGTGTTTTTCTTAGTCCGTTACGTAGGTAGGAATCTGATGTAA4151TGGTTGTTGGTATTTTGTCACCATTCATTTTTATCTGGTTGTTCTCAAGT4201TCGGTTACGAGATCCATTTGTCTATCTAGTTCAACTTGGAAAATCAACGT4251ATCAGTCGGGCGGCCTCGCTTATCAACCACCAATTTCATATTGCTGTAAG4301TGTTTAAATCTTTACTTATTGGTTTCAAAACCCATTGGTTAAGCCTTTTA4351AACTCATGGTAGTTATTTTCAAGCATTAACATGAACTTAAATTCATCAAG4401GCTAATCTCTATATTTGCCTTGTGAGTTTTCTTTTGTGTTAGTTCTTTTA4451ATAACCACTCATAAATCCTCATAGAGTATTTGTTTTCAAAAGACTTAACA4501TGTTCCAGATTATATTTTATGAATTTTTTTAACTGGAAAAGATAAGGCAA4551TATCTCTTCACTAAAAACTAATTCTAATTTTTCGCTTGAGAACTTGGCAT4601AGTTTGTCCACTGGAAAATCTCAAAGCCTTTAACCAAAGGATTCCTGATT4651TCCACAGTTCTCGTCATCAGCTCTCTGGTTGCTTTAGCTAATACACCATA4701AGCATTTTCCCTACTGATGTTCATCATCTGAGCGTATTGGTTATAAGTGA4751ACGATACCGTCCGTTCTTTCCTTGTAGGGTTTTCAATCGTGGGGTTGAGT4801AGTGCCACACAGCATAAAATTAGCTTGGTTTCATGCTCCGTTAAGTCATA4851GCGACTAATCGCTAGTTCATTTGCTTTGAAAACAACTAATTCAGACATAC4901ATCTCAATTGGTCTAGGTGATTTTAATCACTATACCAATTGAGATGGGCT4951AGTCAATGATAATTACTAGTCCTTTTCCTTTGAGTTGTGGGTATCTGTAA5001ATTCTGCTAGACCTTTGCTGGAAAACTTGTAAATTCTGCTAGACCCTCTG5051TAAATTCCGCTAGACCTTTGTGTGTTTTTTTTGTTTATATTCAAGTGGTT5101ATAATTTATAGAATAAAGAAAGAATAAAAAAAGATAAAAAGAATAGATCC5151CAGCCCTGTGTATAACTCACTACTTTAGTCAGTTCCGCAGTATTACAAAA5201GGATGTCGCAAACGCTGTTTGCTCCTCTACAAAACAGACCTTAAAACCCT5251AAAGGCTTAAGTAGCACCCTCGCAAGCTCGGGCAAATCGCTGAATATTCC5301TTTTGTCTCCGACCATCAGGCACCTGAGTCGCTGTCTTTTTCGTGACATT5351CAGTTCGCTGCGCTCACGGCTCTGGCAGTGAATGGGGGTAAATGGCACTA5401CAGGCGCCTTTTATGGATTCATGCAAGGAAACTACCCATAATACAAGAAA5451AGCCCGTCACGGGCTTCTCAGGGCGTTTTATGGCGGGTCTGCTATGTGGT5501GCTATCTGACTTTTTGCTGTTCAGCAGTTCCTGCCCTCTGATTTTCCAGT5551CTGACCACTTCGGATTATCCCGTGACAGGTCATTCAGACTGGCTAATGCA5601CCCAGTAAGGCAGCGGTATCATCAACAGGCTTACCCGTCTTACTGTCnGG5651ATCGACGCTCTCCCTTATGCGACTCCTGCAT

PKOV(pKO5)16-Jan-19968673 bpBased on pKO3: Link, A.J., Phillips, D. and Church, G.M. (1997) Methods for generating precise deletions and insertions in the genome of wild-typeEscherichia coli: Application to open reading frame characterization. J. Bacteriology 179: 6228-6237. The temperature-sensitive pSC101 replicon is functional at 30 C; not at 43 C; Hamilton et. al. (1989) J. Bacteriology 171: 4617-4622NOTE: This file is constructed from various GenBank files and is not primary sequence and has not been confirmed.PKOV has slight advantages over pKO3 in that the NotI cloning site can beeasily used together with BamHI and because of a stuffer the double-digestcan be cleanly separated from singly cut contaminants by gel. The pKOV cloning site is: 5 - SmaI - NotI - SmaI- stuffer - BamHI - SalI - 3BamHI and SalI are not used together, nor is SmaI used with NotI. BglII & BclI cut ends are compatIBLewithBamHI.PmeI&SwaIarecompatiblewithSmaI.pKOVwaspreviouslynamedpKO5,buthaschangednamesincepKO5andrelatedgalKpromotervectorsarealreadyinVectorDB:http://BIOLOGy.queensu.ca/~miseners/vector_descrip/COMPLETE/PKO5.SEQ.htmlForapplicationsandupdatessee:http://arep.med.harvard.edu***************************pKOVpKO3basecoordinates:221-880221-880Cm-R;chloramphenicolacetyltransferase13-103813-1038Cm-R;region1048,10891048SmaIcloningsite(s)10571057,1088NotIcloningsite(s)1064-2456nonePssRLeftflankingregion2457-2579nonec22"universal"KO-tag2580-3287nonePssRrightflankingregion4079-40851068-1074BamHIcloningsite4091-40971099-1105SalIcloningsite4115-45711119-1575f1(+)filamentousorigin4658-65642129-3550BacillussubtilissacBgene7131-86394139-4215origin(pSC101)7399-83494407-5357RepA(temperature-sensitive)pkovLength:8673May9,199904:58Type:NCheck:2716..CCCTTTCGTCTTCGAATAAATACCTGTGACGGAAGATCACTTCGCAGAATAAATAAATCCTGGTGTCCCTGTTGATACCGGGAAGCCCTGGGCCAACTTTTGGCGAAAATGAGACGTTGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTGGTGCCCTTAAACGCCTGGTGCTACGCCTGAATAAGTGATAATAAGCGGATGAATGGCAGAAATTCGAAAGCAAATTCGACCCGGTCGTCGGTTCAGGGCAGGGTCGTTAAATAGCCGCTTATGTCTATTGCTGGTTTANTCGGTACCCGGGGATCGCGGCCGCCCCGAAACCAACTGATGCAGTATCCCGGGCGCTACAACATGATCTCAGTGATGTTATCGGTCAGGAACAAGGAAAGCGAGGACTGGAAATTACCGCTGCTGGCGGGCACAACCTTTTACTGATTGGGCCGCCGGGAACAGGTAAAACAATGCTCGCCAGCCGTATTAATGGCCTGTTGCCAGATTTAAGCAATGAAGAGGCACTGGAGAGTGCTGCGATATTAAGTCTGGTAAATGCTGAATCAGTACAAAAACAATGGCGGCAGCGCCCGTTCCGCTCACCTCATCACAGTGCATCGTTAACTGCGATGGTAGGCGGTGGCGCAATTCCAGGGCCCGGTGAAATTTCGCTGGCGCATAACGGCGTGCTTTTTCTTGATGAGCTACCTGAATTTGAACGGCGTACACTGGATGCCTTGCGAGAGCCGATTGAATCCGGGCAGATCCATCTTTCACGCACACGAGCAAAAATAACCTATCCAGCCCGTTTCCAGCTTGTTGCGGCGATGAATCCCAGCCCTACCGGACATTATCAGGGAAACCATAACCGCTGCACGCCAGAACAGACATTACGTTATCTCAACCGGCTCTCGGGGCCCTTTCTCGACCGCTTCGATCTCTCACTGGAGATCCCATTACCACCCCCCGGCATTTTGAGTAAAACGGTAGTGCCGGGAGAAAGCAGCGCCACCGTTAAACAACGCGTAATGGCCGCCAGAGAGCGCCAATTTAAGCGGCAGAATAAACTGAATGCCTGGCTGGATAGTCCGGAAATACGCCAATTCTGCAAACTTGAGAGCGAAGATGCGATGTGGTTGGAAGGAACACTGATCCATCTGGGGTTATCGATTCGTGCCTGGCAGCGGTTATTGAAAGTTGCACGAACCATTGCTGATATTGATCAGTCTGACATTATCACACGTCAGCATTTGCAGGAGGCAGTTAGCTATCGAGCGATTGACCGTTTGCTCATCCATCTGCAGAAACTACTGACATAAAAAAAGGGCATTTCGCCCTTTTTATTAATCGTCAGAATCGGTGTAGTCTTCAGCACCTTCAACCTGCGGTTTACCGCGGAAAGGGTGTGAAAACGTTTTGGACGCTTAATACGCGTCATATACTTGGACCACACGCGTTCTGCTTCTGTCACTGGCTCACGTTCGCCACGGCATACTGCTACGAAGAGTTTCTCTTCCTCGGTAACCGGCTCGCGTTTGCCAAGATCCAACTCATTGAAGGCATAACCATGACGCTCAAGCAGTTGTGCCTCTTTGATGGTGAAATCACCATGACGAGAGAATCCACGTGGATAATGTTTATTGTCGAAATATCGATTAGTCGTCGTAAAGCTTTCCGCCATCCTGCACGCTCCTAATTCTTTGACCGAGCTAGTTATGGCGCGGAGTATTAGTTACGCTTGACAGAGTGTAAAACAAAACATTTAAATCATAACGACAAATAATTTTGCGGAGAGCACTGTGGATGTTATAAATTTGGAGTGTGAAGGTTATTGCGTGTGGTAAAACAGCTGCATGAACGCCAGCTTGATCTTCTTATTACCACTGAAGCGCCCAAAATGGACGAATTTAGTAGTCAGTTGCTGGGATATTTCACTTTAGCGCTTTATACCAGTGCCCCTTCAAAACTAAAGGGAGATCTTAATTATCTGCGACTTGAGTGGGGGCCAGATTTTCAACAGCATGAGGCAGGTTTGATCGGTGCTGACGAAGTGCCCATTCTGACAACCAGTTCTGCTGAACTGGCACAGCAACAGATTGCGATGCTTAATGGTTGCACCTGGCTACCCGTCAGCTGGGCGCGTAAAAAAGGCGGCCTGCATACCGTTGTCGATAGCACAACACTTTCACGGCCGCTTTATGCCATATGGCTGCAAAATAGCGATAAAAATGCGTTGATTCGCGATCTTTTGAAAATTAACGTGCTGGATGAAGTGTATTAATATGAATGGCTGGCAAGGATGCCGGTAGAAGGATTTACTTCGGAGAGGGTTATTTCAGATAAAAAAAATCCTTACGTTTCGCTAAGGATGATTTCTGGCAGGGGCGGAGAGACTCGAACTCCCAACACCCGGTTTTGGAGACCGGTGCTCTACCAATTGAACTACGCCCCTAATTAGGGTGGCGGAACGGACGGGACTCGAACCCGCGACCCCCTGCGTGACAGGCAGGTATTCTAACCGACTGAACTACCGCTCCACCGAATTCTTTTACAACCACCGGTTTTATGACCGGCTTACTGCTTAATTTGATGCCTGGCAGTTCCCTACTCTCGCATGGGGAGACCCCACACTACCATCGGCGCTACGGCGTTTCACTTCTGAGTTCGGCATGGGGTCAGGTGGGACCACCGCGCTACGGCCGCCAGGCAAATTCTGTTTCATCAGACCGCTTCTGCGTTCTGATTTAATCTGTATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACATCTTCGGCGTTGTAAGGTTAAGCCTCACGGTTCATTAGTACCGGTTAGCTCAACGCATCGCTGCGCTTACACACCCGGCCTATCAACGTCGTCGTCTTCAACGTTCCTTCAGGACTCTCAAGGAGTCAGGGAGAACTCATCTCGGGGCAAGTTTCGTGCTTAGATGCTTTCAGCACTTATCTCTTCCGCATTTAGCTACCGGGCAGTGCCATTGGCATGACAACCCGAACACCAGTGATGCGTCCACTCCGGTCCTCTCGTACTAGGAGCAGCCCCCCTCAGTTCTCCAGCGCCCACGGCAGATAGGGACCGAACTGTCTCACGACGTTCTAAACCCAGCTCGCGTACCACTTTAAATGGCGAACAGCCATACCCTTGGGACCTACTTCAGCTCCAGGATGTGATGAGCCGACATCGAGGTGCCAAACACCGCCGTCGATATGAACTCTTGGGCGGTATCAGCCTGTTATCCCCGGAGTACCTTTTATCCGTTGAGCGATGGCCCTTCCATTCAGAACCACCGGATCACTATGACCTGCTTTCGCACCTGGGATCCTCTAGAGTCGACCGGNGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATNNCANGATCCTTTTTAACCCATCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATCAAGTTCCTGAATTCGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACGCTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGACCAAACACAAGAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGATTTCTCCGGTAAACATTACGGCAAACAAACACTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAACATCAACGGTGTAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAAACGTATCAAAATGTACAGCAGTTCATCGATGAAGGCAACTACAGCTCAGGCGACAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGGCCACAAATACTTAGTATTTGAAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACAAAGCATACTATGGCAAAAGCACATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAAACGCACGGCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCTAAACGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAACACAGTAACAGATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAAATGGTACCTGTTCACTGACTCCCGCGGATCAAAAATGACGATTGACGGCATTACGTCTAACGATATTTACATGCTTGGTTATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGCTGAACAAAACTGGCCTTGTGTTAAAAATGGATCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCTGTACCTCAAGCGAAAGGAAACAATGTCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCAGACAAACAATCAACGTTTGCGCCAAGCTTCCTGCTGAACATCAAAGGCAAGAAAACATCTGTTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGTTAACAAATAAAAACGCAAAAGAAAATGCCGATNNCCGGTTTATTGACTACCGGAAGCAGTGTGACCGTGTGCTTCTCAAATGCCTCAGGCTGTCTATGTGTGACTGTTGAGCTGTAACAAGTTGTCTCAGGTGTTCAATTTCATGTTCTAGTTGCTTTGTTTTACTGGTTTCACCTGTTCTATTAGGTGTTACATGCTGTTCATCTGTTACATTGTCGATCTGTTCATGGTGAACAGCTTTAAATGCACCAAAAACTCGTAAAAGCTCTGATGTATCTATCTTTTTTACACCGTTTTCATCTGTGCATATGGACAGTTTTCCCTTTGATATCTAACGGTGAACAGTTGTTCTACTTTTGTTTGTTAGTCTTGATGCTTCACTGATAGATACAAGAGCCATAAGAACCTCAGATCCTTCCGTATTTAGCCAGTATGTTCTCTAGTGTGGTTCGTTGTTTTTGCGTGAGCCATGAGAACGAACCATTGAGATCATGCTTACTTTGCATGTCACTCAAAAATTTTGCCTCAAAACTGGTGAGCTGAATTTTTGCAGTTAAAGCATCGTGTAGTGTTTTTCTTAGTCCGTTACGTAGGTAGGAATCTGATGTAATGGTTGTTGGTATTTTGTCACCATTCATTTTTATCTGGTTGTTCTCAAGTTCGGTTACGAGATCCATTTGTCTATCTAGTTCAACTTGGAAAATCAACGTATCAGTCGGGCGGCCTCGCTTATCAACCACCAATTTCATATTGCTGTAAGTGTTTAAATCTTTACTTATTGGTTTCAAAACCCATTGGTTAAGCCTTTTAAACTCATGGTAGTTATTTTCAAGCATTAACATGAACTTAAATTCATCAAGGCTAATCTCTATATTTGCCTTGTGAGTTTTCTTTTGTGTTAGTTCTTTTAATAACCACTCATAAATCCTCATAGAGTATTTGTTTTCAAAAGACTTAACATGTTCCAGATTATATTTTATGAATTTTTTTAACTGGAAAAGATAAGGCAATATCTCTTCACTAAAAACTAATTCTAATTTTTCGCTTGAGAACTTGGCATAGTTTGTCCACTGGAAAATCTCAAAGCCTTTAACCAAAGGATTCCTGATTTCCACAGTTCTCGTCATCAGCTCTCTGGTTGCTTTAGCTAATACACCATAAGCATTTTCCCTACTGATGTTCATCATCTGAGCGTATTGGTTATAAGTGAACGATACCGTCCGTTCTTTCCTTGTAGGGTTTTCAATCGTGGGGTTGAGTAGTGCCACACAGCATAAAATTAGCTTGGTTTCATGCTCCGTTAAGTCATAGCGACTAATCGCTAGTTCATTTGCTTTGAAAACAACTAATTCAGACATACATCTCAATTGGTCTAGGTGATTTTAATCACTATACCAATTGAGATGGGCTAGTCAATGATAATTACTAGTCCTTTTCCTTTGAGTTGTGGGTATCTGTAAATTCTGCTAGACCTTTGCTGGAAAACTTGTAAATTCTGCTAGACCCTCTGTAAATTCCGCTAGACCTTTGTGTGTTTTTTTTGTTTATATTCAAGTGGTTATAATTTATAGAATAAAGAAAGAATAAAAAAAGATAAAAAGAATAGATCCCAGCCCTGTGTATAACTCACTACTTTAGTCAGTTCCGCAGTATTACAAAAGGATGTCGCAAACGCTGTTTGCTCCTCTACAAAACAGACCTTAAAACCCTAAAGGCTTAAGTAGCACCCTCGCAAGCTCGGGCAAATCGCTGAATATTCCTTTTGTCTCCGACCATCAGGCACCTGAGTCGCTGTCTTTTTCGTGACATTCAGTTCGCTGCGCTCACGGCTCTGGCAGTGAATGGGGGTAAATGGCACTACAGGCGCCTTTTATGGATTCATGCAAGGAAACTACCCATAATACAAGAAAAGCCCGTCACGGGCTTCTCAGGGCGTTTTATGGCGGGTCTGCTATGTGGTGCTATCTGACTTTTTGCTGTTCAGCAGTTCCTGCCCTCTGATTTTCCAGTCTGACCACTTCGGATTATCCCGTGACAGGTCATTCAGACTGGCTAATGCACCCAGTAAGGCAGCGGTATCATCAACAGGCTTACCCGTCTTACTGTCNGGATCGACGCTCTCCCTTATGCGACTCCTGCAT

ThesefileshavebeenconstructedfromvariousGenBankfiles,arenotprimarydata,andhavenotbeenconfirmed.