protocol for ez-tn5™ insertion kit

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EZ-Tn5™ <KAN-2> Insertion KitCat. Nos. EZI982K, EZI96K2

www.epicentre.com Lit.#134•8/2012 1 EPILIT134 Rev. A

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EZ-Tn5™ Transposon Tools

1. IntroductionTransposons are mobile DNA sequences found in the genomes of prokaryotes and eukaryotes. Transposon tagging has long been recognized as a powerful research tool for randomly distributing primer binding sites, creating gene “knockouts”, and introducing a physical tag or a genetic tag into large target DNAs. One frequently used transposition system is the Tn5 system isolated from gram-negative bacteria.Though a naturally occurring transposition system, the Tn5 system can be readily adapted for routine use in research laboratories for the following reasons:1) Tn5 transposase is a small, single subunit enzyme that has been cloned and purified

to high specific activity.2) Tn5transposasecarriesouttranspositionwithouttheneedforhostcellfactors.3) Tn5 transposon insertions into target DNA are highly random.4) Tn5 transposition proceeds by a simple “cut and paste” process. Although the

chemistry is unique, the result is similar to using a restriction endonuclease, with random sequence specifi city, accompanied by a DNA ligase activity.

5) Tn5 transposase will transpose any DNA sequence contained between its short 19 basepair Mo saic End (ME) Tn5 transposase recognition sequences.

In1998GoryshinandReznikoff 1 demonstrated that a fully functional Tn5 transposition system could be reconstituted in vitro. Additionally, the transposition efficiency of thissystemhasbeenincreasedmorethan1,000-foldcomparedtowild-typeTn5byintroducing mutations in the transposase gene and in the 19-bp Tn5 ME transposase recognition sequence. Epicentre’s EZ-Tn5 Transposon Tools (kits and reagents) are based onthehyperactiveTn5transpositionsystemdevelopedbyGoryshinandReznikoff.

2. Kit ContentsCat. # Concentration Quantity

Reagentsincludedinthekitaresufficientfor10in vitro transposon insertion reactions.

EZ-Tn5™ Transposase:10 U @1 U/µl 10µlin50%glycerolcontaining50 mMTris-HCl(pH 7.5),0.1 MNaCl,0.1 mMEDTA,1 mMdithiothreitol,and0.1%Triton®X-100.

EZ-Tn5™ <KAN-2> Transposon:[email protected] pmol/µl(0.081 µg/µl) 10µlinTEBuffer(10 mMTris-HCl[pH7.5]and1 mMEDTA).

EZ-Tn5™ 10X Reaction Buffer: 100µl0.50 MTris-acetate(pH7.5),1.5 Mpotassiumacetate,100 mMmagnesiumacetate,and40 mMspermidine.

EZ-Tn5™ 10X Stop Solution: 100µl1%SDSsolution.

KAN-2 FP-1 Forward Primer:1nmol @50µM 20µlinTEBuffer(10 mMTris-HCl[pH7.5]and1 mMEDTA).

KAN-2 RP-1 Reverse Primer:1nmol @50µM 20µlinTEBuffer(10 mMTris-HCl[pH7.5]and1 mMEDTA).

pUC19/3.4 Control Target DNA:1µg @0.1 µg/µl 10µlA 3.4-kb Hpa II fragment of bacteriophage DNA cloned into the Acc IsiteofpUC19, inTEBuffer(10 mMTris-HCl[pH7.5]and1 mMEDTA).

Sterile Water: 1 ml

[email protected]•(800)284-8474 3


3. Related ProductsThe following products are also available:– TransforMax™EC100™ElectrocompetentE. coli •Transformationefficiency>109cfu/µgDNA.– ColonyFast-Screen™Kits– MasterPure™DNAPurificationKits– EZ-Tn5™TnpTransposome™Kits– EZ-Tn5™ Transposase– EZ-Tn5™ Transposons– EZ-Tn5™InsertionKits

How the EZ-Tn5 <KAN-2> Insertion Kit Works.TheEZ-Tn5<KAN-2>InsertionKitcanbeusedtorandomlyinsertprimerbindingsitesand a kana mycin resistance selection marker into target DNA in vitro.Asingle2-hourin vitroreactionrandomlyinsertsthe<KAN-2>TransposonintothetargetDNA.Usean aliquot of the reaction to transform E. colisuchasEpicentre’sTransforMax™EC100™Electrocompetent E. coli strain and select on kanamycin plates. Only those clones harboringDNAcontainingthe<KAN-2>Transposonwillgrow.TheEZ-Tn5<KAN-2>InsertionKitcanbeusedfor:• FastersequencingoflargeDNAmolecules,ascomparedtoprimerwalking,random

subcloning, or generating nested deletions with exonuclease III and mung bean nuclease.

• Makinginsertionmutantsorgene“knockouts”in vitro.• IntroducingakanamycinresistanceselectionmarkerintoanyDNA.

Fig.1describesthestepsinvolvedwhenusingtheEZ-Tn5<KAN-2>InsertionKit.Theprocess can be summarized as follows:

Preparation• Prepare0.2 µgofrecombinantDNAfortheEZ-Tn5<KAN-2>insertionreaction.Day 1• Performthe2-hourin vitroEZ-Tn5<KAN-2>insertionreaction.• TransformcompetentrecA— E. coliwith1 µlofthereactionmix.• Selectforkanamycin-resistanttransposoninsertionclonesonkanamycinplates

overnight.Day 2• PrepareDNAfromkanamycin-resistantcolonies.• (Optional)MaptheEZ-Tn5<KAN-2>Transposoninsertionsites.• (Optional)DNAsequencechosenclonesbidirectionallyusingtheunlabeledforward

and reverse transposon-specific primers supplied in the kit.

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1. Incubate 37oC; 2 hrs2. Transform E. coli3. Select KanR clones

Sequence bidirectionally from primer binding sites ( )

4. Prepare plasmid DNA

EZ-Tn5<KAN-2>

TransposonEZ-Tn5

Transposase

KANRTargetDNA

Figure 1. EZ-Tn5 <KAN-2> Transposon Insertion Protocol.



4. MaterialsStorage

StoreEZ-Tn5InsertionKitsonlyat–20°Cinafreezerwithoutadefrostcycle.Theenzymesolution will not freeze. Other component solutions will freeze. After thawing, be sure to mix the contents of each tube thoroughly before using. After use, return all of the kit componentsto–20°Cforstorage.

Performance Specifications and Quality Control

TheEZ-Tn5<KAN-2>InsertionKitisfunction-testedbyperformanceofthekit’sin vitro control reac tion followed by electroporation into a recA— E. coli host strain having a transformationefficiencyof>109 cfu/µgDNA.Transpositionfrequency,definedastheratioofthenumberofKanR clones divided by the number of transformants resistant to theantibioticmarkerofthetargetvector,(KanRcolonies/AmpR colonies; for the control DNA)mustbe>0.5%(commonlyat10%)andtranspositionefficiencymustbe>106KanR colonies/mgtargetDNA.Primersarefunction-testedinaDNAcyclesequencingreactionusingtheSequiThermEXCEL™IIDNASequencingKitandinaPCRreactionusingaplasmidcontaininganEZ-Tn5<KAN-2>Transposonastemplate.AllcomponentsoftheEZ-Tn5<KAN-2>InsertionKitarefreeofdetectableDNaseandRNaseactivitiesasjudgedby agarose gel electro phoresis following over-digestion assays, with the exception of the inherent endonucleolytic function of the EZ-Tn5 Transposase.

5. Transposon Insertion ReactionTarget DNA Preparation

The target DNA must not contain a kanamycin resistance gene. The transposon insertion reaction is not significantly inhibited by high levels of RNA contamination in target DNA preparations.However,ifthetargetDNAisheavilycontaminatedwithchromosomalDNA, which is a direct competitor for tar get transposition, the number of clones will be greatly reduced.

Plasmid and cosmid clones can be purified by standard minilysate procedures and used as target DNA in the insertion reaction without further clean-up. Low copy-number vectors, for example BAC or fosmid clones, are often contaminated with a higher molar proportion of E. coli chromosomal DNA thus reducing the transposon insertion frequency. Therefore, BAC and fosmid DNA should be puri fied, to remove the chromosomal DNA prior to the insertion reaction.

In Vitro Transposon Insertion Reaction

Reaction conditions have been optimized to maximize the efficiency of the EZ-Tn5 Transposon inser tion while minimizing multiple insertion events. Be sure to calculate the moles of target DNA used in the reaction and add an equimolar amount of the EZ-Tn5 <KAN-2> Transposon.Ifnecessary,dilutetheEZ-Tn5<KAN-2>TransposonwithTEbuffer(10 mMTris-HCl[pH 7.5],1 mMEDTA).

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1. Prepare the transposon insertion reaction mixture by adding in the following order:

1 µl EZ-Tn510XReactionBuffer 0.2 µgtargetDNA* x µl molarequivalentEZ-Tn5<KAN-2>Transposon x µl sterilewatertoareactionvolumeof9 µl 1 µl EZ-Tn5Transposase

10 µl Totalreactionvolume

*CalculationofµmoletargetDNA:

µmoltargetDNA=µgtargetDNA/[(#basepairsintargetDNA)x660]

Forexample:0.2µgofcontrolpUC19/3.4DNAwhichis6,100bp

=0.2µg/[6,100 bpx660]=0.05x10-6µmol=0.05 pmol

2. Incubatethereactionmixturefor2hoursat37°C.

3. Stopthereactionbyadding1 µlEZ-Tn510XStopSolution. Mixandheatfor10minutesat70°C. Proceed to section 4, Transformation and Recovery or store the reaction mixture at –20°C.

6. Selection of Transposon Insertion ClonesTransformation and Recovery

The number of EZ-Tn5 Transposon insertion clones obtained per reaction depends on, among other factors, the transformation efficiency of the competent cells used. The greater the transformation effi ciency of the competent cells, the greater the number of insertion clones obtained. We recommend using electrocompetent or chemically competent recA— E. coliwithatransformationefficiencyof>108 cfu/µgofDNA.ArecA— strain of E. coli is recommended to eliminate the possibility of generat ing multimeric forms of the vector. Finally, the host strain used must not have a kanamycin resist ance markerwhenusedwiththeEZ-Tn5<KAN-2>Transposon.Epicentre’sTransforMaxEC100Electrocompetent E. coli(availableseparately)haveatransformationefficiencyof>1x109cfu/µgandareidealforthisapplication.

1) Using1 µloftheinsertionreactionmixture,transformrecA— E. coli. If electrocompetent cells are used, perform electroporation according to the equipmentmanufacturer’srecommendedconditions.Useof>1 µlofthetransposoninsertion reaction for transformation may result in arcing. The unused portion of the transposoninsertionreactioncanbestoredat–20°Cforfutureuse.

2) RecovertheelectroporatedcellsbyaddingSOCmediumtotheelectroporationcuvette to 1 ml final volume immediately after electroporation. Pipette the medium/cellsgentlytomix.Transfertoatubeandincubateona37°Cshakerfor30-60minutestofacilitatecelloutgrowth.



Plating and Selecting Transformants

EZ-Tn5<KAN-2>Transposoninsertionclonesareselectedonkanamycin-containingplates,however,thetransposonwillalsoconferresistancetoneomycinandG418inE. coli.

1) Iftransformationwasdoneusingcellswithanefficiencyof>5x108 cfu/µgDNA,itmaybenecessarytodilutethecells1:10or1:100priortoplating.Plateportions(e.g.,100 µl)ofcellsontoLBplatescontaining50 µg/mlkanamycin.Storetheunusedportionoftherecoveredcellsat+4°Cforupto2daysintheeventadditionalplates need to be prepared.

2) (Optional)Todeterminethetransposoninsertionefficiency,plateidenticaldilutionsand dilution aliquots of the transformation reaction on a second plate containing an antibioticspecificforselectingtargetDNA(e.g.,100 µg/mlampicillinforthecontrolDNA).ThetranspositionfrequencyisgivenbytheratioofKanR/AmpR clones for the control DNA.

3) Growplatesovernightat37°C.Assumingatransposoninsertionefficiencyof1%anduseofhighpuritytargetDNA(i.e.,littleornochromosomalDNAcontamination),oneshouldsee100-500KanR clones per plate. If too few (or too many) colonies appear, replate the transformed cells at a lower (or higher) dilution.

TypicalresultsobtainedwiththeEZ-Tn5<KAN-2>InsertionKitare:• Transposoninsertionfrequency=0.5-20%• TranspositionclonesperµgtargetDNA=1x105-1x108

• Transpositionclonesper10 µltransposoninsertionreaction=1x104-1x107

TheactualnumberofEZ-Tn5<KAN-2>insertionclonesobtainedwillvarydependingonfactors such as target DNA size and the transformation efficiency of the competent cells used to recover the trans poson insertion clones.

7. DNA Sequencing of Transposon Insertion ClonesTransposon Insertion Mapping (optional)

EZ-Tn5 Transposon insertion clones can be sequenced bidirectionally using the unlabeled forward and reverse transposon-specific primers provided in the kit. The insertion site of each clone can also be mapped prior to sequencing, depending on the cost-effectivenessandexperienceoftheuser.

EZ-Tn5<KAN-2>TransposoninsertionsitescanbemappedbysizeanalysisofPCRproducts using colony minilysate DNA as a template. To map the insertion sites, use the KAN-2FP-1orKAN-2RP-1primersprovidedwiththekitandavector-specificflankingprimers (not provided).

Alternatively,insertionsitescanbemappedbyrestrictionendonucleasedigest(s).UsethenucleotidesequenceandrestrictioninformationoftheEZ-Tn5<KAN-2>Transposonprovided in the Appen dix for reference.

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Primer Considerations

TheKAN-2FP-1ForwardandKAN-2RP-1ReversePrimerssuppliedwiththekithavebeencon structed to minimize homology to commonly used cloning vectors. However, the sequence of each primer should be compared to that of the user’s specific cloning vector to ensure minimal se quence homology to the vector. The sequence and theoretical melting temperatures for each pri mer are presented in the Appendix.

Note: Occasionally a clone will yield the sequence of the cloning vector. This occurs when the EZ-Tn5 Transposon randomly inserts into a non-essential region of the vector rather than into the DNA insert. The frequency of this occurrence is dependent on the size of the DNA insert relative to the size of non-essential regions of vector. The larger the DNA insert, the less frequently an insertion will occur into the vector.

Target Site Duplication

EZ-Tn5 Transposase-catalyzed transposon insertion results in the generation of a 9-bp targetsitesequenceduplicationwhereonecopyimmediatelyflankseachsideoftheinserted transposon. This is important to consider when assembling the nucleotide sequence of a recombinant clone insert. The process of transposon insertion site duplicationisdepictedinFig. 2.

Distinguishing Transposon Sequence from Insert Sequence

SincetheprimersprovidedintheEZ-Tn5<KAN-2>InsertionKitannealtoaregionnearthe ends of the transposon, the first sequence data obtained from each sequencing reactionisthatofEZ-Tn5<KAN-2>TransposonDNA.Thesequenceofthe19-bpEZ-Tn5Transposaserecognitionsequence(ME)foundatthejunctionofthetransposonandthe target clone insert DNA (present in all insertion clones), is a useful landmark to distinguish vector sequence from target clone insert sequence (see also Fig. 3).

8. TroubleshootingArcing during transformation by electroporation

1) Excessive salt in target DNA preparation.Uselesssampleforelectroporation.Ethanolprecipitateand70%ethanolwash,ordropdialyzeDNA.

Arcing inevitably results in failed transformation. Discard the electroporation reactionandtryagainwith0.5 µlofthetranspositionreaction.WiththecontrolDNA,noarcingisobservedwhenupto2 µloftranspositionreactionisusedforelectroporationof50 µlofelectrocompetentrecA— E. coliina0.2-cmwidthelectroporationcuvetteandusinganEppendorfMultiporatorat2500 V.Avoltagegradientofapproximately12,500 V/cmisfairlystandardforE. coli.

No, or few transposition clones on selective plates1) Transformation reaction was unsuccessful; low competence. Test by plating

outgrowth using drug resistance marker on target DNA to distinguish between transformation or transposon inser tion failure.



9 b.p. Staggered Cut

Target DNA5'3'

A B C D E F G H I

A B C D E F G H I ' ''''''''

9 b.p. Gaps5'3' A B C D E F G H I ' ''''''''

A B C D E F G H I

MEME

+EZ-Tn5™ TRANSPOSASE

EZ-Tn5™TRANSPOSON

MEME

E. coli DNA Repair after Transformation

5'3'

A B C D E F G H I

A B C D E F G H I ' ''''''''A B C D E F G H I

A B C D E F G H I ' ''''''''

MEME9 b.p.Direct RepeatTarget Site Duplication

Transesterification Reaction

Random Transposon Insertion

5'

3' GACAGAGAATATGTGTAGACTGTCTCTTATACACATCT AGATGTGTATAAGAGACAG

TCTACACATATTCTCTGTCEZ-Tn5™ TRANSPOSON

19 bp Mosaic End Sequence19 bp Mosaic End Sequence

ForwardPrimer

ReversePrimer

9 b.p. Staggered Cut

Target DNA5'3'

A B C D E F G H I

A B C D E F G H I ' ''''''''

9 b.p. Gaps5'3' A B C D E F G H I ' ''''''''

A B C D E F G H I

MEME

+EZ-Tn5™ TRANSPOSASE

EZ-Tn5™TRANSPOSON

MEME

E. coli DNA Repair after Transformation

5'3'

A B C D E F G H I

A B C D E F G H I ' ''''''''A B C D E F G H I

A B C D E F G H I ' ''''''''

MEME9 b.p.Direct RepeatTarget Site Duplication

Transesterification Reaction

Random Transposon Insertion

5'

3' GACAGAGAATATGTGTAGACTGTCTCTTATACACATCT AGATGTGTATAAGAGACAG

TCTACACATATTCTCTGTCEZ-Tn5™ TRANSPOSON

19 bp Mosaic End Sequence19 bp Mosaic End Sequence

ForwardPrimer

ReversePrimer

Figure 2. EZ-Tn5 Transposase Insertion Site Duplication Process.

Figure 3. EZ-Tn5 Transposon Insertion Site Junction.

Ifcompetentcellshaveatransformationefficiency<108 cfu/µgDNA,onemaynot obtain suffi cient clones on a plate. For example, transforming into cells with transformationefficiency<105 cfu/µgDNAresultsinasfewas2insertionclonesonaplate.Usecellswithatransformationefficiency>108 cfu/µgDNA.

2) Transposon insertion reaction was unsuccessful. Inhibitor contamination in target DNA. Pur ify target DNA further. Perform procedure with control plasmid provided with kit to assure system components are functional.

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DNA sequencing results are ambiguous

1) Two or more transposon insertions into target clone. Discard the clone. Choose other clone(s) to sequence.

The protocol was designed to minimize multiple transposon insertion events. Even so,about1%ofthetranspositionclonesmaycontain>1transposon.Onecanverifysingle insertion clones by agarose gel electrophoresis of colony minilysates prior to sequencing.Asingleinsertionshouldbeabout1.2 kblargerthantheparentaltargetDNA.Adoubleinsertionwillincreasethesizeby2.4 kbandalsoresultin“doublesequence”.

2) The sequencing primer used has significant homology to the cloning vector or to the DNA being sequenced. Check homology of primer against the vector. Alter primer annealing condi tions or synthesize a new primer with less homology.

3) Components of the DNA sequencing kit and/or of the electrophoresis step are compromised. Reverifytheintegrityofthecomponentsofthekitand/orelectrophoresisstepwithappropriatecontrols.Useanewkitand/ornewreagents.

Confluent plates following transformation1) Target DNA or host cells have the same selective marker (antibiotic resistance)

as the transposon used.Useadifferenthostfortransformationandretransformwith a portion of your remaining reaction.

SomehostcellscarryKanR transposons. Confirm that the genotype of the host strain chosenforthetransformationisnotKanR.

9. Appendix

Primer DataKAN-2 FP-1 Forward Primer

5′ - ACCTACAACAAAGCTCTCATCAACC - 3′

Length:25nucleotidesG+C content: 11

Molecular Weight:7,484daltonsTemperatures of Dissociation & Melting:Td: 68°C (nearestneighbormethod)

Tm: 73°C (%G+Cmethod)

Tm: 72°C ([2(A+T)+4(G+C)]method)

Tm: 63°C ((81.5+16.6(log[Na+]))+([41(#G+C)-500]/length)method) where[Na+]=0.1M



Figure 4. EZ-Tn5 <KAN-2> Transposon.

EZ-Tn5 <KAN-2> Transposon Schematic Map

EZ-Tn5™ <KAN-2> Transposon(1,221 bp)

KAN-2 FP-1 Forward PrimerKAN-2 RP-1 Reverse PrimerME = Mosaic End

5' ACCTACAACAAAGCTCTCATCAACC 3'5' GCAATGTAACATCAGAGATTTTGAG 3'5' AGATGTGTATAAGAGACAG 3'

KANR

BspH I 87Xho I 167

Nru I 226

Nci I 442Ssp I 494

Pvu I 569Hae I 656

PflM I 832

Ava II 1122BamH I 1159

Xba I 1165Sal I 1171

Acc I 1172Hinc II 1173

Pst I 1181Sph I 1187

Hind III 1189

ME MEATG138

TAA953 KAN-2 FP-1

1127-1151KAN-2 RP-1

43-67

Note: Not all restriction enzymes that cut only once are indicated above.

Primers are not drawn to scale.See the following pages for further information.

1 100 200 300 500 700 900 1221400 600 800 1000 1100

KAN-2 RP-1 Reverse Primer

5′ - GCAATGTAACATCAGAGATTTTGAG - 3′

Length:25nucleotidesG+C content: 9Molecular Weight:7,705daltonsTemperatures of Dissociation & Melting:Td: 65°C (nearestneighbormethod)

Tm: 69°C (%G+Cmethod)

Tm: 68°C ([2(A+T)+4(G+C)]method)

Tm: 60°C ((81.5+16.6(log[Na+]))+([41(#G+C)-500]/length)method) where[Na+]=0.1M



EZ-Tn5 <KAN-2> Transposon Restriction Data

Restriction Enzymes that cut the EZ-Tn5 <KAN-2> Transposon one to three times:

Enzyme Sites LocationAccI 1 1172AciI 1 174AluI 2 1139,1191ApoI 2 182,366AsiSI 1 569AseI 1 768AvaI 1 167AvaII 1 1122BamHI 1 1159BanII 1 224Bfa I 1 1166BfrBI 2 417,683BfuAI 1 1184Bpu10I 1 586BsaWI 1 704BsiE I 1 569BsmI 2 453,530BsmBI 1 585Bsp1286I 1 224BspDI 2 29,260BspHI 1 87BspMI 1 1184BsrI 3 360,984,1124BsrD I 1 61BsrFI 1 523BssKI 3 440,457,814BstF5I 2 200,826BstNI 2 459,816BstUI 3 176,226,571BstYI 2 818,1159BtgI 2 1089,1150BtsI 2 430,517ClaI 2 29,260DdeI 2 586,1041DsaI 2 1089,1150EarI 1 382EcoNI 1 481FauI 1 1148

Enzyme Sites Location Fnu4HI 2 174,1013HaeI 1 656HaeIII 2 173,656HincII 1 1173HindIII 1 1189HpaII 3 442,524,705HpyCH4IV 1 159Mae II 1 159MboII 3 369,480,1058MlyI 2 802,1178MspI 3 443,525,706MwoI 3 281,313,527NciI 1 442NlaIV 1 1161NruI 1 226NsiI 2 419,685NspI 1 1187PaeR7I 1 167PflMI 1 832PleI 2 801,1177PspGI 2 457,814PstI 1 1181Pvu I 1 569RsaI 1 404SalI 1 1171Sau96I 1 1122SbfI 1 1181ScrFI 3 442,459,816SfcI 1 1177SmlI 1 167SphI 1 1187SspI 1 494TliI 1 167Tsp45I 1 716TspRI 3 442,517,989XbaI 1 1165XhoI 1 167



Restriction Enzymes that cut the EZ-Tn5 <KAN-2> Transposon four or more times:

Alw IBsaJ IBsl IBsmA ICac8I

CviJ IDpn IHhaIHinfIHinPI

HphIHpy188IHpyCH4IIIHpyCH4VMae III

Mbo IMnl IMse INla IIISau3AI

SfaNITaq ITfi ITsp4C ITsp509I

Aat IIAcc65 IAcl IAfe IAflIIAflIIIAge IAhd IAle IAlwN IApa IApaB IApaL IAsc IAvr IIBan IBbs IBbvC IBciVIBcl IBgl IBgl II

Blp IBme1580IBmgB IBmr IBsa IBsaA IBsaB IBsaHIBseY IBsiHKAIBsiW IBspE IBspLU11IBsrB IBsrGIBssHIIBssSIBstAP IBstB IBstE IIBstXIBstZ17I

Bsu36 IDra IDra IIIDrd IEae IEag IEco47IIIEcoO109IEcoR IEcoRVFse IFsp IGdiIIHaeIIHpaIHpy99IKpnIMfe IMlu IMsc IMsl IMspA1 I

Nae INar INco INde INgoMIVNhe INot IPac IPci IPflFIPme IPml IPpuM IPshA IPsi IPspOM IPvu IIRsr IISacISacIISanDISapI

ScaISexAISfiISfoISgrAISimISmaISnaBISpeISrfISse8647IStuIStyISwaITat ITse ITth111 IXcmIXmaIXmnI

Restriction Enzymes that do not cut the EZ-Tn5 <KAN-2> Transposon:



EZ-Tn5 <KAN-2> Transposon Sequence

EZ-Tn5™ <KAN-2> Transposon 1,221 bp.

1 CTGTCTCTTA TACACATCTC AACCATCATC GATGAATTGT GTCTCAAAAT

51 CTCTGATGTT ACATTGCACA AGATAAAAAT ATATCATCAT GAACAATAAA

101 ACTGTCTGCT TACATAAACA GTAATACAAG GGGTGTTATG AGCCATATTC

151 AACGGGAAAC GTCTTGCTCG AGGCCGCGAT TAAATTCCAA CATGGATGCT

201 GATTTATATG GGTATAAATG GGCTCGCGAT AATGTCGGGC AATCAGGTGC

251 GACAATCTAT CGATTGTATG GGAAGCCCGA TGCGCCAGAG TTGTTTCTGA

301 AACATGGCAA AGGTAGCGTT GCCAATGATG TTACAGATGA GATGGTCAGA

351 CTAAACTGGC TGACGGAATT TATGCCTCTT CCGACCATCA AGCATTTTAT

401 CCGTACTCCT GATGATGCAT GGTTACTCAC CACTGCGATC CCCGGAAAAA

451 CAGCATTCCA GGTATTAGAA GAATATCCTG ATTCAGGTGA AAATATTGTT

501 GATGCGCTGG CAGTGTTCCT GCGCCGGTTG CATTCGATTC CTGTTTGTAA

551 TTGTCCTTTT AACAGCGATC GCGTATTTCG TCTCGCTCAG GCGCAATCAC

601 GAATGAATAA CGGTTTGGTT GATGCGAGTG ATTTTGATGA CGAGCGTAAT

651 GGCTGGCCTG TTGAACAAGT CTGGAAAGAA ATGCATAAAC TTTTGCCATT

701 CTCACCGGAT TCAGTCGTCA CTCATGGTGA TTTCTCACTT GATAACCTTA

751 TTTTTGACGA GGGGAAATTA ATAGGTTGTA TTGATGTTGG ACGAGTCGGA

801 ATCGCAGACC GATACCAGGA TCTTGCCATC CTATGGAACT GCCTCGGTGA

851 GTTTTCTCCT TCATTACAGA AACGGCTTTT TCAAAAATAT GGTATTGATA

901 ATCCTGATAT GAATAAATTG CAGTTTCATT TGATGCTCGA TGAGTTTTTC

951 TAATCAGAAT TGGTTAATTG GTTGTAACAC TGGCAGAGCA TTACGCTGAC

1001TTGACGGGAC GGCGGCTTTG TTGAATAAAT CGAACTTTTG CTGAGTTGAA

1051GGATCAGATC ACGCATCTTC CCGACAACGC AGACCGTTCC GTGGCAAAGC

1101AAAAGTTCAA AATCACCAAC TGGTCCACCT ACAACAAAGC TCTCATCAAC

1151CGTGGCGGGG ATCCTCTAGA GTCGACCTGC AGGCATGCAA GCTTCAGGGT

1201TGAGATGTGT ATAAGAGACA G

ThetransposonsequencecanbedownloadedattheURL:http://www.epicentre.com/sequences



10. ReferencesCited:

1. Goryshin,I.Y.andReznikoff,W.S.(1998)J. Biol. Chem. 273,7367.

Development of the hyperactive EZ-Tn5 (Tn5) in vitro transposition system:

1. Goryshin,I.Y.andReznikoff,W.S.(1998)J. Biol. Chem. 273,7367.2. Zhou,M.et al.,(1998)J. Mol. Biol. 276, 913.

3. Zhou,M.andReznikoff,W.S.(1997)J. Mol. Biol. 271,362.4. Mahnke-Braam,I.A.andReznikoff,W.S.(1998)J. Biol. Chem. 273,10908.

These products are intended for research use only.

EZ-Tn5™ Transposon Tools for in vitro transposon insertion are covered by U.S. Patent Nos. 5,925,545; 5,948,622; 5,965,443, and 6,437,109; European Patent No. 0927258, and other pat ents issued or pending, exclusively licensed or assigned to Epicentre. These products are accom-panied by a limited non-exclusive license for the purchaser to use the purchased product(s) solely for in vitro transposon insertion for life science research. Purchase of these products does not grant rights to: (1) offer products, components of products, or any derivatives thereof for resale; or (2) to distribute or transfer the products, components of products, or any derivatives thereof to third parties. Contact Epicentre for information on licenses for uses other than life science research.

Triton is a registered trademark of Rohm & Haas, Philadelphia, Pennsylvania.

EC100, EZ-Tn5, Fast-Screen, TransforMax, MasterPure, SequiTherm EXCEL, and Transposome are trademarks of Epicentre, Madison, Wisconsin.

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