Surface-sterilized leaf discs or other axenic plants are infected with the appropriate strain of A. tumefaciens, and cocultured on regeneration medium for 2 or 3 days. During this time, the virulence genes in the bacteria are induced, the bacteria bind to the plant cells around the wounded edge of the explant, and the gene transfer process occurs. After the transformation has occurred, the explants are transferred to regeneration/selection medium. This contains 500 ug/ ml carbenicillin to kill the bacteria and the appropriate antibiotics (usually kanamycin) to inhibit untransformed plant cells. During the next 3-4 weeks, the transformed cells grow into callus or differentiate into shoots via organogenesis. Between 3 and 6 weeks, the shoots develop enough to remove them from the explant and induce rooting in preparation for transfer to soil.

MATERIALS AND SOLUTIONS

MS-G Medium (1 liter)
MS Salts (Gibco) ———————————– 4.3 g
B5 Vitamin Stock ———————————- 1 ml
3% Sucrose —————————————- 30 g
0.8% Agar —————————————— 8 g
Add deionized H₂O to make a final volume of —– 1 liter

15. Fertilize and grow under standard plant growth conditions.

NOTES

KIT INFORMATION
REFERENCES

3 ml 1 M Tris-HCl pH 7.5

+ 150µl 2 M MgCl₂

+ 240 µl 100 mM dNTP mix (25 mM each: dGTP, dCTP, dATP, dTTP)

+ 300µl 1 M DTT

+ 1.5 g PEG-8000

+ 300µl 100 mM NAD

+ dH20 to 6 ml

Store at -20 oC in 320 µl aliquots.

2) Prepare 0.5  ml of Gibson Cloning master mix as follows:

320 µl 5X ISO Buffer

+ 0.64 µl 10 U/µl T5 exonuclease*

+ 20 µl 2 U/µl Phusion polymerase

+ 160 µl 40 U/µl Taq ligase

DO NOT ADD WATER

Store at -20 oC in 5 µl aliquots.This is 3.2 X, This mix allows you to add sample and water to 16 µl

This is optimized for 20-150 bp sequence homology overlaps

3) Thaw a 5 µl aliquot of the Gibson assembly master mix, and keep on ice until use.

4) Measure the DNA concentration (ng/µl) of each assembly piece.

5) Add 100 ng of the linearized vector backbone and equimolar amounts of the other assembly pieces in 11µl (e.g., elute DNA from spin column with 11 µl water after gel purification) to the thawed 5µl master mix in a 16 µl total volume assembly reaction mixture as follows:

linearized vector backbone (100 ng) + each additional assembly piece (to equimolar with backbone)==11  µl

+ 5 µl Gibson Cloning assembly master mix

6) Incubate the assembly reaction at 50 oC for 15 ~ 60 minutes, and then place on ice.

        70         80         90        100        110        120
PEPIQLLNAK QILGQLDGGS VAVLPVIDST NQYLLDRIGE LKSGDACIAE YQQAGRGRRG

       130        140        150        160        170        180
RKWFSPFGAN LYLSMFWRLE QGPAAAIGLS LVIGIVMAEV LRKLGADKVR VKWPNDLYLQ

       190        200        210        220        230        240
DRKLAGILVE LTGKTGDAAQ IVIGAGINMA MRRVEESVVN QGWITLQEAG INLDRNTLAA

       250        260        270        280        290        300
MLIRELRAAL ELFEQEGLAP YLSRWEKLDN FINRPVKLII GDKEIFGISR GIDKQGALLL

       310        320
EQDGIIKPWM GGEISLRSAE K
AviTag:  GLNDIFEAQKIEWHE.
ggtggcggtctgaacgacatcttcgaggctcagaaaatcgaatggcacgaa
 G  G  G  L  N  D  I  F  E  A  Q  K  I  E  W  H  E

- – not active
+ – active
na – not applicable
1 – strand displacement activity of Pfu DNA Polymerase is temperature-dependent. It will perform strand displacement at temperatures higher than 60°C.

Sequence

MATTKTSGMALALLLVAALA
VGAAAWGKEGHRLTCMVAEP
FLSSESKQAVEELLSGRDLP
DLCSWADQIRRSYKFRWTGP
LHYIDTPDNLCTYDYDRDCH
DSHGKKDVCVAGGINNYSSQ
LETFLDSESSSYNLTEALLF
LAHFVGDIHQPLHVAFTSDA
GGNGVHVRWFGRKANLHHVW
DTEFISRANRVYYHDISKML
RNITRSITKKNFNSWSRCKT
DPAACIDSYATESIDASCNW
AYKDAPDGSSLDDDYFSSRL
PIVEQRLAQGGVRLASILNR
IFGGAKSNRSSRSSM*

Met Ala Thr Thr Lys Thr Ser Gly Met Ala Leu Ala Leu Leu Leu Val
Ala Ala Leu Ala Val Gly Ala Ala Ala Trp Gly Lys Glu Gly His Arg  
Leu Thr Cys Met Val Ala Glu Pro Phe Leu Ser Ser Glu Ser Lys Gln
Ala Val Glu Glu Leu Leu Ser Gly Arg Asp Leu Pro Asp Leu Cys Ser
Trp Ala Asp Gln Ile Arg Arg Ser Tyr Lys Phe Arg Trp Thr Gly Pro
Leu His Tyr Ile Asp Thr Pro Asp Asn Leu Cys Thr Tyr Asp Tyr Asp
Arg Asp Cys His Asp Ser His Gly Lys Lys Asp Val Cys Val Ala Gly
Gly Ile Asn Asn Tyr Ser Ser Gln Leu Glu Thr Phe Leu Asp Ser Glu
Ser Ser Ser Tyr Asn Leu Thr Glu Ala Leu Leu Phe Leu Ala His Phe
Val Gly Asp Ile His Gln Pro Leu His Val Ala Phe Thr Ser Asp Ala
Gly Gly Asn Gly Val His Val Arg Trp Phe Gly Arg Lys Ala Asn Leu
His His Val Trp Asp Thr Glu Phe Ile Ser Arg Ala Asn Arg Val Tyr
Tyr His Asp Ile Ser Lys Met Leu Arg Asn Ile Thr Arg Ser Ile Thr
Lys Lys Asn Phe Asn Ser Trp Ser Arg Cys Lys Thr Asp Pro Ala Ala
Cys Ile Asp Ser Tyr Ala Thr Glu Ser Ile Asp Ala Ser Cys Asn Trp
Ala Tyr Lys Asp Ala Pro Asp Gly Ser Ser Leu Asp Asp Asp Tyr Phe
Ser Ser Arg Leu Pro Ile Val Glu Gln Arg Leu Ala Gln Gly Gly Val
Arg Leu Ala Ser Ile Leu Asn Arg Ile Phe Gly Gly Ala Lys Ser Asn
Arg Ser Ser Arg Ser Ser Met

Cel-1: Cel-1 can be purified from Celery juice. Ready to use kits can be purchased from Transgenomic, Inc.

SURVEYOR Mutation Detection Kit For Standard Gel Electrophoresis
Catalog No. Quantity
706025	  25 Reactions, $79
706020	100 Reactions, $265
706021	1,000 Reactions, $2050
Phone: 888-813-7253

http://www.transgenomic.com

1. Run PCR gene synthesis reaction on an agarose gel, purify desired band using Qiagen gel extraction kit. Elute DNA with 50 ul TE.

1. Denature purified DNA at 95 oC for 10 min, anneal at 0.1 oC per second for 10 min in a buffer containing 100 mM Tris-HCl, pH 8.0, 1 mM EDTA and 1 M NaCl.

2. Purify DNA using Qiagen PCR purification kit.

3. Add buffer (10 mM Tris-HCl, pH 8.8, 75 mM KCl, 1.5 mM MgSO4) to DNA in 45 ul, add 5 ul of mis-match specific endonuclease CEL1 nuclease from Transgenomic, Inc. Incubate at 40 oC for 60 min.

4. Run digested DNA on agarose gel, cut out "error-free" CEL1 resistant band and purify DNA using Qiagen gel extraction kit

Cel-1 Sequence

MTRLYSVFFL LLALVVEPGV RAWSKEGHVM TCQIAQDLLE PEAAHAVKML LPDYANGNLS
        70         80         90        100        110        120
SLCVWPDQIR HWYKYRWTSS LHFIDTPDQA CSFDYQRDCH DPHGGKDMCV AGAIQNFTSQ
       130        140        150        160        170        180
LGHFRHGTSD RRYNMTEALL FLSHFMGDIH QPMHVGFTSD MGGNSIDLRW FRHKSNLHHV
       190        200        210        220        230        240
WDREIILTAA ADYHGKDMHS LLQDIQRNFT EGSWLQDVES WKECDDISTC ANKYAKESIK
       250        260        270        280        290
LACNWGYKDV ESGETLSDKY FNTRMPIVMK RIAQGGIRLS MILNRVLGSS ADHSLA

Cel-1 like s1 nucleases can be extracted and purified from these plants

[Apium graveolens]

http://en.wikipedia.org/wiki/Celery
 [Petroselinum crispum]

http://en.wikipedia.org/wiki/Parsley

 [Solanum lycopersicum]

http://en.wikipedia.org/wiki/Tomato
[Zinnia elegans]

http://en.wikipedia.org/wiki/Zinnia_elegans
[Solanum tuberosum]

http://en.wikipedia.org/wiki/Potato

 [Vitis vinifera]

http://en.wikipedia.org/wiki/Vitis_vinifera

[Humulus lupulus]

http://en.wikipedia.org/wiki/Humulus_lupulus
 [Glycine max]

http://en.wikipedia.org/wiki/Soybean

 [Hemerocallis hybrid cultivar]

http://en.wikipedia.org/wiki/Daylily

 [Sorghum bicolor]

http://en.wikipedia.org/wiki/Sorghum_bicolor
[Brachypodium distachyon]

http://en.wikipedia.org/wiki/Brachypodium_distachyon

 [Spinacia oleracea]

http://en.wikipedia.org/wiki/Spinach

[Arabidopsis lyrata subsp. lyrata]

http://en.wikipedia.org/wiki/Arabidopsis_lyrata

 [Zea mays]

http://en.wikipedia.org/wiki/Maize

 [Medicago truncatula]

http://en.wikipedia.org/wiki/Medicago_truncatula

 [Arabidopsis thaliana]

http://en.wikipedia.org/wiki/Arabidopsis_thaliana

[Ricinus communis]

http://en.wikipedia.org/wiki/Castor_oil_plant

 [Oryza sativa ]

http://en.wikipedia.org/wiki/Oryza_sativa
 [Populus trichocarpa]

http://en.wikipedia.org/wiki/Populus_trichocarpa
 [Dianthus caryophyllus]

http://en.wikipedia.org/wiki/Dianthus_caryophyllus
 [Cucumis sativus]

http://en.wikipedia.org/wiki/Cucumber
 [Picea sitchensis]

http://en.wikipedia.org/wiki/Picea_sitchensis
 [Zinnia violacea]

http://en.wikipedia.org/wiki/Zinnia
 [Physcomitrella patens subsp. patens]

http://en.wikipedia.org/wiki/Physcomitrella_patens

[Hordeum vulgare subsp. vulgare]

http://en.wikipedia.org/wiki/Barley

[Selaginella moellendorffii]

http://en.wikipedia.org/wiki/Selaginella_moellendorffii
 [Brachypodium distachyon]

http://en.wikipedia.org/wiki/Brachypodium_distachyon
 

A list of some plant S1 nucleases
>CEL I mismatch endonuclease [Apium graveolens]
1 mtrlysvffl llalvvepgv rawskeghvm tcqiaqdlle peaahavkml lpdyangnls
       61 slcvwpdqir hwykyrwtss lhfidtpdqa csfdyqrdch dphggkdmcv agaiqnftsq
      121 lghfrhgtsd rrynmteall flshfmgdih qpmhvgftsd mggnsidlrw frhksnlhhv
      181 wdreiiltaa adyhgkdmhs llqdiqrnft egswlqdves wkecddistc ankyakesik
      241 lacnwgykdv esgetlsdky fntrmpivmk riaqggirls milnrvlgss adhsla
>PARS I endonuclease [Petroselinum crispum]
   1 mtrlysvffl llalvvepgv rawskeghvm tcqiaqdlle peaahavkml lpdyangnls
       61 slcvwpdqir hwykyrwtss lhfidtpdqa csfdyqrdch dphggedmcv agaiqnftsq
      121 lghfrhgtsd rrynmteall flshfmgdih qpmhvgftsd mggnsidlrw frhksnlhhv
      181 wdreiiltaa adyhgkdmhs llqdiqrnft egswlqhves wkecddistc ankyakesik
      241 lacnwgykdv esgetlsdky fntrmpivmk riaqggirls milnrvlgss adhsla
>endonuclease 1 [Solanum lycopersicum]
     1 mlrltslssi fflcvafinq hgveawskeg hvmtcriaqg llndeaahav kmllpeyvng
       61 dlsalcvwpd qvrhwykykw tsplhfidtp dkacnfdyer dchdqhgvkd mcvagaiqnf
      121 ttqlshyreg tsdrrynmte allflshfmg dihqpmhvgf tsdaggnsid lrwfrhksnl
      181 hhvwdreiil taakdyyakd inlleedieg nftdgiwsdd laswrecgnv fscvnkfate
      241 siniackwgy kgveagetls ddyfnsrlpi vmkrvaqggi rlamllnnvf gasqqedsvv
      301 at
>endonuclease precursor-like precursor [Solanum lycopersicum]
 1 mlrltllssi fflcvafinq hgveawskeg hvmtcriaqg llndeaahav kmllpeyvng
       61 dlsalcvwpd qvrhwykykw tsplhfidtp dkacnfdyer dchdqhgvkd mcvagaiqnf
      121 ttqlshyreg tsdrrynmte allflshfmg dihqpmhvgf tsdaggnsid lrwfrhksnl
      181 hhvwdreiil taakdyyakd inlleedieg nftdgiwsdd laswrecgnv fscvnkfate
      241 siniackwgy kgveagetls ddyfnsrlpi vmkrvaqggi rlamllnnvf gasqqedsvv
      301 at
>endonuclease [Zinnia elegans]
        1 malirlsiis clgffminny navqawskeg hvmtcqiaqe llspdaahav qmllpdyvkg
       61 nlsalcvwpd qirhwyryrw tsplhfidtp ddacsfdytr dchdsngmvd mcvagaiknf
      121 tsqlshyqhg tsdrrynmte allfvshfmg dihqpmhvgf ttdeggntid lrwfrhksnl
      181 hhvwdreiil taaselydkd meslqkaiqa nfthglwsdd vnswkdcddi sncvnkyake
      241 sialackwgy egveagetls ddyfdsrmpi vmkriaqggv rlsmilnrvf gssssledal
      301 vpt
>endonuclease [Solanum tuberosum]
        1 mlrltllsii fflclafinh hgaeawskeg hmmtcriaqg llndeaahav kmllpeyvng
       61 dlsalcvwpd qvrhwykykw tsplhfidtp dkacnfdyer dchdqhgvkd mcvagaiqnf
      121 ttqlshyreg tsdrrynmte allflshfmg dihqpmhvgf tsdaggnsid lrwfrhksnl
      181 hhvwdreiil taakdyyakd vnlleedieg nftdgiwsdd laswrecgnv fscvnkfate
      241 siniackwgy kgveagetls ddyfnsrlpi vmkrvaqggi rlamllsnvf gvsqqedsvg
      301 at
>PREDICTED: nuclease S1 [Vitis vinifera]
        1 mgkfgglfrl srsgfgllvv ftfvmapgal awskeghimt cqiaqallep eaaeavrnll
       61 pdyvngdlsa lctwpdqirh wykyrwtssl hfidtpdeac tfdysrdchd phglqdmcva
      121 gaiknftsql shygegssdr rynlteallf lshfmgdihq plhvgftsde ggntielhwf
      181 rhksnlhhvw dreiiltaaa dfytkdmdll ledikgnftd gvwaddvssw kecndlltcp
      241 nkyasesisi ackwgykgak pgstladeyf nsrmpiamkr iaqggvrlsm ilnrvfghth
      301 gkmsaat
>Vitis vinifera
        1 mapgalawsk eghimtcqia qallepeaae avrnllpdyv ngdlsalctw pdqirhwyky
       61 rwtsslhfid tpdeactfdy srdchdphgl qdmcvagaik nftsqlshyg egssdrrynl
      121 teallflshf mgdihqplhv gftsdeggnt ielhwfrhks nlhhvwdrei iltaaadfyt
      181 kdmdllledi kgnftdgvwa ddvsswkecn dlltcpnkya sesisiackw gykgakpgst
      241 ladeyfnsrm piamkriaqg gvrlsmilnr vfghthgkms aat
>pre-endonuclease [Humulus lupulus
        1 mdrysfglvl vlalasitvp sahgwskegh tmtcqiaqal ldseaaeavr nllpdyvngd
       61 lsalcvwpdq vrhwyryrwt splhfidtpd qacsfdysrd chdqhglenm cvagaiqnft
      121 sqlshyregt sdrrynmtea llflshfmgd ihqplhvgft tdeggntinl rwfrhksnlh
      181 hvwdreiilt alkdyyeknt dlllqdikgn ytdgvwsddv sswehcddla scinkfaves
      241 iniackwgyk dvepgvtlad dyfdsrmpil mkriaqggvr lamilnrvlg esdeglatpt

>nuclease PA3-like [Glycine max]
     1 mvrlgrlwms llpfgfllff ssafirvpga lawskeghvm tcqiaqalle peaseavyql
       61 lpdyvkgnls slciwpdqir hwykyrwtsp lhfidtpdna csfqysrdch dshgvedmcv
      121 agavknftsq lmhykegtsd rrynmteall flshfmgdih qpmhvgfttd eggntielhw
      181 frhksnlhhv wdreiiltal adyydkdvsl llqdiernyt dgiwsddits wkhcndisqc
      241 vnnwakesiq iackwgyegv eagatladdy fdsrvpfvmk riaqggirla milnkvfads
      301 eerfatat
>SP I mismatch endonuclease [Spinacia oleracea]
        1 msrstcfvsc lvycliliag vqawskeghm itckiaqdll epeaadavkk llpedlngnl
       61 salcvwpdqv rhwykyrwts plhfidtpdd scsfdykrdc hdehgvedmc vagaiqnfts
      121 qlqhyrqgta drrynlteal lflshfmgdi hqpmhcahts drggnsidlr wfrhksnlhh
      181 vwdreiilta mkdyydndvd llqqdivgnf tdgiwfddvs hwtdcdslht cvtkyatesi
      241 niackwgykd adpgvtlgdn yfnsrlpfvm kriaqggvrl amilnkvfnh gheamvvat
>nuclease PA3-like [Brachypodium distachyon]
        1 mrsrwpmdsa afllavvlvs apvarswste ghmltcqiaq dllepaaada vknllpeeag
       61 gdlsalcvwp dqvrhwykyk wssplhfidt pdqacsfvys rdchdpsgak dmcvagaian
      121 ftsqlmhykh gsadrkynlt eallflshfm gdvhqpmhvg ftsdqggnsv nlrwfkhksn
      181 lhhvwdreii ltalaehygk dmdafrkdle hniskgawsd disswgdcqd llscptkyat
      241 esislackwg yssvhdgdtl sddyfasrlp ivarriaqgg vrlamilnrv fgesnrdvla
      301 wpnlaaatde l
>Sorghum bicolor
        1 matvsllraa alglvvlasa lpaarswske ghmltcqiaq dllepdaaha vrnllpddvg
       61 gdlsalcvwp dqvrhwykyk wtgplhfidt pdkacsfdys rdchgpdgak dmcvagaian
      121 ftsqllhykh gsadrrynlt eallflshfm gdvhqpmhvg ftsdqggnsi elrwfrhksn
      181 lhhvwdreii qtaladfygk dmdafrkqle hnltkgtwsd dvsawtdced lsscptkyat
      241 esielackwa ysgvregetl sddyfdsrlp ivsrriaqgg vrlamflnri fgahnrdvpp
      301 aaaaas
>Arabidopsis lyrata subsp. lyrata
        1 masafrsstr lipvlgilil csvssvrsws keghiltcri aqnlleagpa hvvenllpdy
       61 vkgdlsalcv wpdqirhwyk yrwtshlhyi dtpdqacsye ysrdchdqhg lkdvcvdgai
      121 qnftsqlqhy gegtsdrryn mteallflsh fmgdihqpmh vgftsdeggn tidlrwykhk
      181 snlhhvwdre iiltalkesy dknldllqed leknittglw hddlsswtec ndliacphky
      241 asesiklack wgykgvksge tlseeyfntr lpivmkrivq ggvrlamiln rvfsddhaia
      301 gvaat
>Zea mays

        1 maavsllraa alglvvlasa lpsarpwske ghvltcqiaq gllepdaaha vrnllpddag
       61 gdlsalcvwp dqvrhwyrym wtgplhfidt pdeacsfdys rdchgpdgak dmcvagaian
      121 ftsqllhyrh gsadrrynlt eallflshfm gdvhqpmhvg ftsdqggnsi elrwfrhksn
      181 lhhvwdreii qtaladfygk dmdafrkqle hnltkgtwsd dvsawtdcqd lsscptkyat
      241 esiglackwa ysgvregetl sddyfdsrlp ivsrriaqgg vrlamflnri fgahnrdvpp
      301 pss
>Endonuclease [Medicago truncatula]
        1 marlkglmls lpfgfvlflc stfsivpgvi gwskeghemt cliaqallkp easeavhhll
       61 pphvngnlsa lcvwpdqirh wykyrwtspl hfidtpdekc gfqysrdcle dmcvagaikn
      121 ftsqlshyke gtsdrrynmt eallflshfm gdihqpmhvg ftsdkggnti dlrwyrhksn
      181 lhhvwdreii ltaladyydk dvtlllqdie knytngiwsd dvaswehckd isscvnnwak
      241 esiqiackwg yegvksgmtl sekyfdsrmp yvmkriaqgg irlamilnqv fgdseegfva
      301 at
>bifunctional nuclease 1 [Arabidopsis thaliana]
        1 masafrsstr lilvlgilil csvssvrsws keghiltcri aqnlleagpa hvvenllpdy
       61 vkgdlsalcv wpdqirhwyk yrwtshlhyi dtpdqacsye ysrdchdqhg lkdmcvdgai
      121 qnftsqlqhy gegtsdrryn mteallflsh fmgdihqpmh vgftsdeggn tidlrwykhk
      181 snlhhvwdre iiltalkeny dknldllqed leknitnglw hddlsswtec ndliacphky
      241 asesiklack wgykgvksge tlseeyfntr lpivmkrivq ggvrlamiln rvfsddhaia
      301 gvaat
>endonuclease 4 [Arabidopsis thaliana]
       1 mssslrqwfa rvlvltqlin galcwgkegh ytvckiaesy feeetvaavk kllpksadgd
       61 lasvcswpde ikhhwqwrwt splhyvdtpd yrcnyeycrd chdthknqdr cvtgaifnyt
      121 mqlmsasens dtivhynlte almflshfig dihqplhvgf lgdeggntit vrwyrrktnl
      181 hhvwdnmiie salktyynks lplmiealqa nltndwsndv plwescqlnq tacpnpyase
      241 sinlackyay rnatpgttlg ddyflsrlpi vekrlaqggi rlaatlnrif sskpkhags
>endonuclease 2 [Arabidopsis thaliana]
       1 manqkglhvv mmiitvwlly aapnihgwgk egheiickia qtrldetaak avkellpesa
       61 egdlsslclw adrvkfryhw ssplhyintp dacsyqynrd ckdesgekgr cvagaiynyt
      121 tqllsyktaa ssqsqynlte allfvshfmg dihqplhvsy asdkggntie vhwytrkanl
      181 hhiwdsniie taeadlynsa legmvdalkk nittewadqv krwetctkkt acpdiyaseg
      241 iqaacdwayk gvtegdtled eyfysrlpiv yqrlaqggvr laatlnrifg
>endonuclease 5 [Arabidopsis thaliana]
        1 mrlwivsvlv lthlvhgalc wgkdghytvc klaegffedd tiaavkkllp esvdggglad
       61 fcswpdeikk lsqwqwtstl hyvntpeyrc nyeycrdchd thkhkdwcvt gaifnytnql
      121 msasensqni vhynlteall flshymgdvh qplhtgflgd lggntiivnw yhnksnlhhv
      181 wdnmiidsal etyynsslph miqalqaklk ngwsndvpsw kschfhqkac pnlyasesid
      241 lackyayrna tpgttlgdey flsrlpvvek rlaqggirla atlnrifsak pklagl
>  endonuclease 3 [Arabidopsis thaliana]
        1 mgwslrmwiv silvltqlvn galcwgdagh yavckiaqsy feedtvvavk kllpesange
       61 laavcswpde ikklpqwrwt salhfadtpd ykcnyeysrd cpkdwcvtga ifnytnqlms
      121 tsensqsivh ynltealmfl shymgdihqp lhegfigdlg gnkikvhwyn qetnlhrvwd
      181 dmiiesalet yynsslprmi helqaklkng wsndvpswes cqlnqtacpn pyasesidla
      241 ckyayrnata gttlgdyyfv srlpvvekrl aqggirlagt lnrifsakrk lara
>endonuclease precursor [Fourraea alpina]
        1 masafrsstt lilvfgvlil csvspvqsws keghiltcri aqnlleagpa hvvenllpdy
       61 vkgdlsalcv wpdqirhwyk yrwtsslhyi dtpdeacsye ysrdchdqhg ledmcvdgai
      121 qnftsqlqhy gegtsdrrhn mteallflsh fmgdihqpmh vgftsdeggn tidlrwyrhk
      181 snlhhvwdre iiltalkeyy dkdldllqed leknitngfw dddlsswtec idliacphky
      241 asesiklack ygyegvksge tlsedyfntr mpivmkrivq ggvrlamiln rifsdthagv
      301 aatcg
>Endonuclease [Medicago truncatula]
        1 marlkglmls lpfgfvlflc stfsivpgvi gwskeghemt cliaqallkp easeavhhll
       61 pphvngnlsa lcvwpdqirh wykyrwtspl hfidtpdekc gfqysrdcle dmcvagaikn
      121 ftsqlshyke gtsdrrynmt eallflshfm gdihqisfis yqpmhvgfts dkggntidlr
      181 wyrhksnlhh vwdreiilta ladyydkdvt lllqdiekny tngiwsddva swehckdiss
      241 cvnnwakesi qiackwgyeg vksgmtlsek yfdsrmpyvm kriaqggirl amilnqvfgd
      301 seegfvaat
>Nuclease PA3, putative [Ricinus communis]
        1 mlvsfasifv pgalgwskeg himtcriaqn llgpeaahav ehllphnvng dlsalcvwad
       61 qirhwykyrw tsslhfidtp dkactfdysr dceedrcvag aiqnftsqll hykegstdrr
      121 ynlteallfl shfvgdihqp lhvgftsdeg gntidlrwyr hksnlhhvwd reiiltalkd
      181 yyendmnllq qaiegnftdg iwyddvsswk dcsdilscpn kyaaesisia ckwgykgvkg
      241 gatlsddyfn srmpivmkri aqggirlamf lnqifgdsee giaspt

>Oryza sativa Indica Group
        1 masdkcssfl laaaaaavlv lasapvahsw skeghmltcr iaqdllepaa ahavrnllte
       61 eadgdlsalc vwpdqvrhwy kyrwtsplhf idtpdkacsf vysrdchgad gaedmcvaga
      121 ianftsqlmh ynhgsadrky nmteallfls hfmgdvhqpm hvgftsdqgg ntinlrwfrh
      181 ksnlhhvwdr emiltaiaef ygkdmdafqk dlvhnfttgt wsddvsswgd cedllscstk
      241 yatesinlac kwayndvreg etlsddyfgs rlpivtrria qggvrlamfl nrlfgehnrd
      301 vaspa
>Populus trichocarpa
        1 mdrirgllrl calafiffag lasislpgal gwskeghiit criaqnllgp eaahavenll
       61 phnlngdlsa lciwpdqirh wyryrwtspl hfidtpdkac tfdysrdcvk dacvdgaiqn
      121 ftsqllhyrd gtadrrynlt eallflshfm gdihqpmhvg ftsdeggntv elrwfrhksn
      181 lhhvwdreii ltalkdfydn dmvllqeaie gnftdgiwfd dvaswkdcdd llscpdkyat
      241 esinmackwg ykgvkesvtl addyfdsrmp ivmkriaqgg vrlamflnri fgdpeegfas
      301 pt
>putative nuclease [Dianthus caryophyllus]
        1 vyclifivgv rgwskeghti tcqiaqnlle pealhavknl ipehlngnls alcvwpdqvr
       61 hwykyrwtsp lhfidapdds csfnydrdch dshgvkdmcv agaiqnytsq lqhyregssd
      121 rrynmteall flshfmgdih qpmhcghttd eggntielrw fkhksnlhhv wdreiiltam
      181 kdyydkdvtl lqqdiegnft dgiwandvss wtdceslhac vtkyatesin iackwgykgv
      241 epgvtlsdey fntrlpfvmk ria
>bifunctional nuclease precursor [Cucumis sativus]
        1 mgklvflvvl ifisfllvlp caqgwskegh iltceiaqel lipeaaeavq dllpesaggn
       61 lsamcvwpdq irlqskyrwa splhyantpd scsfvykrdc hndagqpdmc vagairnftt
      121 qlttyrtqgf dsphnlteal lflshfvgdi hqplhvgfes daggntievr wfrrksnlhh
      181 vwdrdiilea lgdyydkdgg llldelnrnl tqgiwsndvs ewercstvns cvnrwadest
      241 glackwayeg veagitlsee yydsrlpivm erlaqggvrl amllnrvfae datrgfayss
>Glycine max
        1 mdrlrrlwms llpfgfllff ssafigvpga lawskeghvm tcqiaqalle peaseavyql
       61 lpdhvkgnls alctwpdqir hwykyrwtsp lhfidtpdna csfqysrdch dpqgvedmcv
      121 agavknftsq lihykegtsd rrynmteall flshfmgdih qpmhvgfttd eggntielrw
      181 frhksnlhhv wdreiiltgl adyydkdvsf llqdiernyt dgiwsddvts wnivmislsv
>Dianthus caryophyllus
      1 pdqvrhwyky rwtsplhfid apddscsfny drdchdshgv kdmcvagaiq nytsqlqhyr
       61 egssdrrynm teallflshf mgdihqpmhc ghttdeggnt ielrwfkhks nlhhvwdrei
      121 iltamkdyyd kdvtllqqdi egnftdgiwa ndvsswtdce slhacvtkya tesiniackw
      181 gykgvepgvt lsdeyfntrl pfvmkriaqg gvrlanpyd
>Picea sitchensis
        1 mrnysrnlss allasvmiyl smvptseswg keghyatcki aqpllseeas aavkkllpdy
       61 aegdlaslcs wadqvrfryr wasplhfidt pdnkctyiys rdchnpegee gmcvdgaikn
      121 ytsqlgnygc ratganynlt eallflshfm gdihqplhvg fstdeggnti klhwygrqnn
      181 lhhiwdtlii etamkdnynn dleemiaaiq knvtdswsde vptwekcstn alacpklyad
      241 esinlacnwa ykdadqnsvl eddyffsrlp ivetqlakgg vrlaatlnri fnst
>bifunctional nuclease [Zinnia violacea]
        1 lslvllllvf vapvtvrgwg vdghfitcki aqgrlsqtav davnsllpey aegdlaslcs
       61 wadhvkfryh wssalhyidt pdnlctyqyr rdckdedgvm grcvagaimn yttqlldygk
      121 qtsqynltea llflshfmgd ihqplhvgft sdrggntidv hwftrkavlh hvwddsiiet
      181 aeerfygsnv enlidaietn itnvwgdqvk awencsanqk tcpniyateg ikaacnwayk
      241 gvtngsvled dyflsrlpiv nwrlaqggvr laanlnrifg
>Hordeum vulgare subsp. vulgare
        1 mapprprpqp allllllvsl laasaprrad awgkeghimv ckvaerylse daaaavqell
       61 pesaggelst vcpwadtmrf ryhwasplhy antpnvcnfd fsrdchnsrg eqgmcvvgai
      121 nnytdqlyty gdssskssyn lteslmflah fvgdvhqplh vgyeedeggn titvhwyrrk
      181 anlhhvwdvs iidtvmkdfy nksldtmvda lqtnltegws ddvgrwesca nkkatcandy
      241 aiesinlacn yaykdvvqni tlgddyyltr ypvvekrlaq agvrlaliln rifdkkkada
      301 mplyiq
>Physcomitrella patens subsp. patens

        1 vlcvatlprr agawgadghh aacliaepll tpttwkavns llperangsl aslctwaddi
       61 kwmwkyhwta plhyidtpdf lcrydydrdc hdqhgqkgfc asgainnfts qltnyelpka
      121 srplltkthn lteallflah ivgdihqplh vgftsdaggn titvhwyrrk tnlhhiwdte
      181 mivkaedvyy nkslsnmvda iilnitnnwi geaqlwgecp kgeiacpdty aaesiklacq
      241 yayrnatpgs tladeyflsr lpivetrlaq agvrlaailn rlfdtk
>nuclease S1-like [Brachypodium distachyon]
        1 maplllvlll aaavsplpap ahgwgvdghl micqiaqsrl sgaaaaavka llprdaggnl
       61 ssvcswadnv rfryhwsapl hfidtpdnlc gysydrdckd ndgvkgrcva gainnytsql
      121 ltygsssshs ssaqynltqa llflshfmgd ihqplhvgft sdrggntidv hwykrktvlh
      181 hvwdasiiet aeddyydrgt aefvdaltkn itgewsekvq gweecsknqt acpdiyases
      241 itaacdwayk nakedstlgd ayfgsrlpvv slrlaqggvr laatlnrifg etkta

>Selaginella moellendorffii
        1 malcvilias iavgaaawgr eghrltcmia epflsseska aleellpgrd lpelcswadd
       61 irrsyrfrwt gplhyidtpd nlcgydydrd chdsrgekdm cvagainnys rqletfqeak
      121 lslhksynlt eallflahyv gdihqplhva ftadaggngv hvhwfgrkan lhhiwdtefi
      181 drakrlyyhd ifrmlrnism sitenfdaws rcetdpeaci dsyatesiht scrwaykdal
      241 egtyleddyf ssrlpiveqr laqggvrlas ilnrifgaks sessi
>Selaginella lepidophylla
MATTKTSGMALALLLVAALAVGAAAWGKEGHRLTCMVAEPFLSSESKQAVEELLSGRDLPDLCSWADQIRRSYKFRWTGP
LHYIDTPDNLCTYDYDRDCHDSHGKKDVCVAGGINNYSSQLETFLDSESSSYNLTEALLFLAHFVGDIHQPLHVAFTSDA
GGNGVHVRWFGRKANLHHVWDTEFISRANRVYYHDISKMLRNITRSITKKNFNSWSRCKTDPAACIDSYATESIDASCNW
AYKDAPDGSSLDDDYFSSRLPIVEQRLAQGGVRLASILNRIFGGAKSNRSSRSSM

Alignment with Arabidopsis S1 nucleases
Cel-1                        -------MTRLYSVFFLLLALVVEPGVR--AWSKEGHVMTCQIAQDLLEP 41
bifunctional_nuclease_1      MASAFRSSTRLILVLGILILCSVSS-VR--SWSKEGHILTCRIAQNLLEA 47
endonuclease_5               --------MRLWIVSVLVLTHLVHGAL---CWGKDGHYTVCKLAEGFFED 39
endonuclease_3               ----MGWSLRMWIVSILVLTQLVNGAL---CWGDAGHYAVCKIAQSYFEE 43
endonuclease_4               ----MSSSLRQWFARVLVLTQLINGAL---CWGKEGHYTVCKIAESYFEE 43
endonuclease_2               ----MANQKGLHVVMMIITVWLLYAAPNIHGWGKEGHEIICKIAQTRLDE 46
Res-1                        ----MATTKTSGMALALLLVAALAVGAA--AWGKEGHRLTCMVAEPFLSS 44
                                          .  ::    :        *.. **   * :*:  :. 

Cel-1                        EAAHAVKMLLPDYANG-NLSSLCVWPDQIRHWYKYRWTSSLHFIDTPDQA 90
bifunctional_nuclease_1      GPAHVVENLLPDYVKG-DLSALCVWPDQIRHWYKYRWTSHLHYIDTPDQA 96
endonuclease_5               DTIAAVKKLLPESVDGGGLADFCSWPDEIKKLSQWQWTSTLHYVNTPEYR 89
endonuclease_3               DTVVAVKKLLPESANG-ELAAVCSWPDEIKKLPQWRWTSALHFADTPDYK 92
endonuclease_4               ETVAAVKKLLPKSADG-DLASVCSWPDEIKHHWQWRWTSPLHYVDTPDYR 92
endonuclease_2               TAAKAVKELLPESAEG-DLSSLCLWADRVK--FRYHWSSPLHYINTPD-A 92
Res-1                        ESKQAVEELLS----GRDLPDLCSWADQIRRSYKFRWTGPLHYIDTPDNL 90
                              .  .*: **.    *  *. .* *.*.::   :::*:. **: :**:  

Cel-1                        CSFDYQRDCHDPHGGKDMCVAGAIQNFTSQLGHFRHGTSDR-RYNMTEAL 139
bifunctional_nuclease_1      CSYEYSRDCHDQHGLKDMCVDGAIQNFTSQLQHYGEGTSDR-RYNMTEAL 145
endonuclease_5               CNYEYCRDCHDTHKHKDWCVTGAIFNYTNQLMSASENSQNIVHYNLTEAL 139
endonuclease_3               CNYEYSRDCP-----KDWCVTGAIFNYTNQLMSTSENSQSIVHYNLTEAL 137
endonuclease_4               CNYEYCRDCHDTHKNQDRCVTGAIFNYTMQLMSASENSDTIVHYNLTEAL 142
endonuclease_2               CSYQYNRDCKDESGEKGRCVAGAIYNYTTQLLSYKTAASSQSQYNLTEAL 142
Res-1                        CTYDYDRDCHDSHGKKDVCVAGGINNYSSQLETFLDSESSS--YNLTEAL 138
                             *.::* ***      :. ** *.* *:: **       .    **:****

Cel-1                        LFLSHFMGDIHQPMHVGFTSDMGGNSIDLRWFRHKSNLHHVWDREIILTA 189
bifunctional_nuclease_1      LFLSHFMGDIHQPMHVGFTSDEGGNTIDLRWYKHKSNLHHVWDREIILTA 195
endonuclease_5               LFLSHYMGDVHQPLHTGFLGDLGGNTIIVNWYHNKSNLHHVWDNMIIDSA 189
endonuclease_3               MFLSHYMGDIHQPLHEGFIGDLGGNKIKVHWYNQETNLHRVWDDMIIESA 187
endonuclease_4               MFLSHFIGDIHQPLHVGFLGDEGGNTITVRWYRRKTNLHHVWDNMIIESA 192
endonuclease_2               LFVSHFMGDIHQPLHVSYASDKGGNTIEVHWYTRKANLHHIWDSNIIETA 192
Res-1                        LFLAHFVGDIHQPLHVAFTSDAGGNGVHVRWFGRKANLHHVWDTEFISRA 188
                             :*::*::**:***:* .: .* *** : :.*: .::***::**  :*  *

Cel-1                        AADYHGKDMHSLLQDIQRNFTEGSWLQDVESWKECD-DISTCANKYAKES 238
bifunctional_nuclease_1      LKENYDKNLDLLQEDLEKNITNGLWHDDLSSWTECN-DLIACPHKYASES 244
endonuclease_5               LETYYNSSLPHMIQALQAKLKN-GWSNDVPSWKSCHFHQKACPNLYASES 238
endonuclease_3               LETYYNSSLPRMIHELQAKLKN-GWSNDVPSWESCQLNQTACPNPYASES 236
endonuclease_4               LKTYYNKSLPLMIEALQANLTN-DWSNDVPLWESCQLNQTACPNPYASES 241
endonuclease_2               EADLYNSALEGMVDALKKNITT-EWADQVKRWETCT-KKTACPDIYASEG 240
Res-1                        NRVYY-HDISKMLRNITRSITK----KNFNSWSRCKTDPAACIDSYATES 233
                                 :   :  :   :  .:.     .:.  *  *  .  :* . **.*.

Cel-1                        IKLACNWGYKDVESGETLSDKYFNTRMPIVMKRIAQGGIRLSMILNRVLG 288
bifunctional_nuclease_1      IKLACKWGYKGVKSGETLSEEYFNTRLPIVMKRIVQGGVRLAMILNRVF- 293
endonuclease_5               IDLACKYAYRNATPGTTLGDEYFLSRLPVVEKRLAQGGIRLAATLNRIFS 288
endonuclease_3               IDLACKYAYRNATAGTTLGDYYFVSRLPVVEKRLAQGGIRLAGTLNRIFS 286
endonuclease_4               INLACKYAYRNATPGTTLGDDYFLSRLPIVEKRLAQGGIRLAATLNRIFS 291
endonuclease_2               IQAACDWAYKGVTEGDTLEDEYFYSRLPIVYQRLAQGGVRLAATLNRIFG 290
Res-1                        IDASCNWAYKDAPDGSSLDDDYFSSRLPIVEQRLAQGGVRLASILNRIFG 283
                             *. :*.:.*:..  * :* : ** :*:*:* :*:.***:**:  ***:: 

Cel-1                        SSADHSLA----- 296
bifunctional_nuclease_1      -SDDHAIAGVAAT 305
endonuclease_5               AKPKLAGL----- 296
endonuclease_3               AKRKLARA----- 294
endonuclease_4               SKPKHAGS----- 299
endonuclease_2               -------------
Res-1                        GAKSNRSSRSSM- 295

Alternatively, design gene sequence using IDT's web site, https://www.idtdna.com/pages/products/genes/custom-gene-synthesis, it is also free

Design 500 to 800 bp fragments for oligo synthsis, for example Taq Ploymerase can be made by gene synthesis of 4 PCR fragments

http://www.ncbi.nlm.nih.gov/nuccore/155128

atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac
181 cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg
241 gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac
301 gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg
361 tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag
421 gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac
481 gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc
541 gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg
601 tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc
661 gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg
721 gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac
781 ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag
841 ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa
901 aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc
961 ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc
     1021 ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat
     1081 cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa
     1141 gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc
     1201 ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg
     1261 gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag
     1321 gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt
     1381 gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc
     1441 ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc
     1501 ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac
     1561 cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt
     1621 cccgccatcg gcaagacgga gaagaccggc aagcgctcca ccagcgccgc cgtcctggag
     1681 gccctccgcg aggcccaccc catcgtggag aagatcctgc agtaccggga gctcaccaag
     1741 ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc
     1801 cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac
     1861 ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc
     1921 gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc
     1981 cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg
     2041 gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgccgg
     2101 gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag
     2161 gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc
     2221 cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg
     2281 gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg
     2341 cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc
     2401 atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc
     2461 cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc
     2521 cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag
     2581 gtggggatag gggaggactg gctctccgcc aaggagtga

Nde1
CATATG

ATGAGGGGGATGCTGCCCCTCTTTGAGCCCAAGGGCCGGGTCCTCCTGGTGGACGGCCACCACCTGGCCTACCGCACCTTCCACGCCCTGAAGGGCCTCACCACCAGCCGGGGGGAGCCGGTGCAGGCGGTCTACGGCTTCGCCAAGAGCCTCCTCAAGGCCCTCAAGGAGGACGGGGACGCGGTGATCGTGGTCTTTGACGCCAAGGCCCCCTCCTTCCGCCACGAGGCCTACGGGGGGTACAAGGCGGGCCGGGCCCCCACGCCGGAGGACTTTCCCCGGCAACTCGCCCTCATCAAGGAGCTGGTGGACCTCCTGGGGCTGGCGCGCCTCGAGGTCCCGGGCTACGAGGCGGACGACGTCCTGGCCAGCCTGGCCAAGAAGGCGGAAAAGGAGGGCTACGAGGTCCGCATCCTCACCGCCGACAAAGACCTTTACCAGCTCCTTTCCGACCGCATCCACGTCCTCCACCCCGAGGGGTACCTCATCACCCCGGCCTGGCTTTGGGAAAAGTACGGCCTGAGGCCCGACCAGTGGGCCGACTACCGGGCCCTGACCGGGGACGAGTCCGACAACCTTCCCGGGGTCAAGGGCATCGGGGAGAAGACGGCGAGGAAGCTTCTGGAGGAG
(GGAGAAGACGGCGAGGAAGCTTCTGGAGGAG)TGGGGGAGCCTGGAAGCCCTCCTCAAGAACCTGGACCGGCTGAAGCCCGCCATCCGGGAGAAGATCCTGGCCCACATGGACGATCTGAAGCTCTCCTGGGACCTGGCCAAGGTGCGCACCGACCTGCCCCTGGAGGTGGACTTCGCCAAAAGGCGGGAGCCCGACCGGGAGAGGCTTAGGGCCTTTCTGGAGAGGCTTGAGTTTGGCAGCCTCCTCCACGAGTTCGGCCTTCTGGAAAGCCCCAAGGCCCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCCCGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTG
(GAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTG)AGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAACATCCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGATAGAGCTCAGGGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTC
(CCTCTCCGGCGACGAGAACCTGATCCGGGTC)TTCCAGGAGGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTCGGGGTCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGGAGCTAGCCATCCCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAAGAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGCTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGTGA

Sal1
GTCGAC

2. Design Oligos for each fragment using NIH DNAWorks software http://helixweb.nih.gov/dnaworks/

Copy and paste DNA sequence to the website, use 50 to 60 nt, do not need to change any other parameters
Select the oligo design (TRIAL) with the lowest Score (with as few # of misprime as possible).
Make sure there are 30 nt overlaps between each PCR fragment.

3. Order oligos from IDT (on plate, should be 8 cents per base).

4. Make oligo stock solution and mixture
Mix: take 1 ul from each 10 uM oligo stock in water.

5. Template generation PCR: 5 cycles using Phusion hot start DNA polymerase or Kapa HiFi
0.5 ul oligo mix
0.5 ul polymerase enzyme
5 ul of 10X buffer
1 ul of 10 mM dNTP
Water to 25 ul

6. PCR

mix two PCR reactions, add two outer oligo and continue PCR 25 cycles.

7. Run PCR product on gel, purify using Qiagen gel extraction kit

8. Join small fragments with Gibson mix.

9. Purify assembled product on gel.

10. Digest with restriction enzyme and clone into a vector.

11. Verify clone by sequencing (Macrogen, $3.5 per reaction).
 

Prepare the following reaction mixture
a) Purified DNA in water 32 μl
b) 10X buffer 5 μl
c) 1 mM dATP 10 μl
d) Klenow Exo- 3 μl
total volume 50 μl

Incubate in a heat block at 37°C for 30 minutes.

Protocol 2

Purified DNA 44µl

5 µl 10X Taq buffer

1 μL 10 mM dATP

0.5 µL Taq polymerase. 

incubate at 72°C for 30 minutes

Other solubility enhancer: maltose-binding protein (MBP), thioredoxin (TRX), transcription pausing
factor L (NusA), thiol-disulfide oxidoreductase, glutathione
S-transferase (GST)

50 min at 70 oC

5 min at 90 oC

120 min at 90 oC

60 min at 95 oC

30 min at 100 o C.

50 min at 95 oC

40 min at 100 oC

35 min at 105 oC

17.29 (TteSSB2)

16.96 (TteSSB3)

Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany: http://www.dsmz.de/catalogues.html

yes

* 1-5% of wild type exonuclease activity is present in the modified
enzyme