genomics & proteomics core facility gateway full orf clone manual · 2019-03-11 · genomics...

Genomics & Proteomics Core Facility 1/31

Bernhard Korn/SW Gateway Full ORF Clone Manual 29.08.2014

Gateway® Full ORF Clone Manual

Ver. Date Name Comment 1.0 27.12.06 Bernhard Korn Basic protocols for handling Gateway Full ORF Clones

1.1 29.08.14 Stefan Wiemann Map and sequence of pDONR223 added

Content

Gateway® Full ORF Clone Manual................................................................................................................1 1. Introduction ..................................................................................................................................................3 2. Flow Chart ....................................................................................................................................................3 3. Quality control at DKFZ ................................................................................................................................4 4. Reagents to be supplied by the User ...........................................................................................................4 5. Protocols ......................................................................................................................................................5

5.1. Streaking stab cultures and preparing strains .......................................................................................5 5.2. Plasmid preparation by alkaline lysis ....................................................................................................6 5.3. Other plasmid preparation methods ......................................................................................................7 5.4. Restriction digest of Gateway Full ORF Clone plasmids ......................................................................7 5.5. Shuttling full ORFs by Gateway® LR reaction ......................................................................................8

5.5.1. Fast Gateway® LR protocol............................................................................................................8 5.5.2. High Confidence Gateway® LR protocol ........................................................................................9 5.5.3. Multi Destination Gateway® LR protocol ..................................................................................... 10

5.6. Preparation of chemically competent E. coli ...................................................................................... 11 5.7. Transformation of Gateway® LR reactions ........................................................................................ 11

6. Appendix ................................................................................................................................................... 12 6.1. Gateway Full ORF Clones: Vector Information .................................................................................. 12

6.1.1. pDONR201 features .................................................................................................................... 12 6.1.2. pDONR201 map .......................................................................................................................... 13 6.1.3. pDONR221 features .................................................................................................................... 14 6.1.4. pDONR221 map .......................................................................................................................... 15 6.1.5. pDONR223 features .................................................................................................................... 16 6.1.6. pDONR223 map .......................................................................................................................... 16

.......................... 16 6.2. Genotypes of E. coli ........................................................................................................................... 17 6.3. Sequences .......................................................................................................................................... 17

6.3.1. Primers ......................................................................................................................................... 17 6.3.2. Vectors ......................................................................................................................................... 18

7. Troubleshooting ........................................................................................................................................ 26 8. Literature ................................................................................................................................................... 28

8.1. General ............................................................................................................................................... 28 8.2. Applications ........................................................................................................................................ 28

8.2.1. Expression in Bacteria ................................................................................................................. 28 8.2.2. Yeast Expression ......................................................................................................................... 29



8.2.3. Baculo Virus Expression .............................................................................................................. 29 8.2.4. Expression in Plant Cells ............................................................................................................. 30 8.2.5. Mammalian Cell Line Expression ................................................................................................ 30 8.2.6. Miscellaneous .............................................................................................................................. 30



1. Introduction

The Gateway Full ORF Clones are designed to provide maximum flexibility. The Backbone of the clones are Gateway® entry vectors (pDONR201 or pDONR221), allowing rapid and efficient transfer the ORF of interest to any destination vector. The insert of each clone is fully sequenced and analyzed. Features of pDONR201 and pDONR221 vectors are:

• attL1 and attL2 sites for site-specific recombination of the Gateway Full ORF Clone with any destination vector of interest when doing an LR reaction

• rrnB transcription termination sequences to prevent read-through and basal expression of the gene of interest in E. coli

• Kozak-like consensus sequence at the start ATG for efficient translation initiation in eukaryotic systems

• Kanamycin resistance gene for selection in E. coli • pUC origin for high-copy replication and maintenance of the plasmid in E. coli

2. Flow Chart

Streak stab culture

(protocol 5.1)

Picking of colonies and preparation of bacterial strains (protocol 5.1)

Minipreps of plasmids (protocols 5.2 and 5.3)

Restriction analysis (protocol 5.4)

Shuttling inserts (Gateway® LR reaction, protocols 5.5.1 – 5.5.3)

(Transformation, protocol 5.7)

Expression construct



3. Quality control at DKFZ

Each clone is grown overnight in LB medium containing 50 µg/ml kanamycin at 37°C. All clones exhibited growth under these conditions. The insert of each clone is sequenced completely, including the vector–insert junctions. For detailed information on the sequence of the clone, please visit www.dkfz.de/gpcf/index.php?id=38 or check for the EMBL/GenBank entry of your clone of interest. Some of the clones do have base substitution relative to RefSeq. These variations are annotated, but you should verify this information by aligning the clone sequence with your desired protein coding sequence. 4. Reagents to be supplied by the User

• Gateway® LR Clonase™ Enzyme Mix 20 reactions, Invitrogen #11791-019 • Destination vector of choice, can either be obtained from Invitrogen, or be constructed by

the user. GPCF will share Gateway destination vectors upon request. The destination vector must contain ccdB gene3 flanked by attR1 and attR2 sites. Verify that the reading frame of the destination vector is compatible with the Gateway Full ORF Clones

• Agar plates containing the antibiotic coded by the destination vector of choice • Transformation-competent E. coli (e.g. XL series, DH series or similar)

http://www.dkfz.de/gpcf/index.php?id=38



5. Protocols

5.1. Streaking stab cultures and preparing strains

We recommend that you prepare bacterial strains of your Gateway Full ORF Clones, to be able to go back to the original master to produce more plasmid DNA if necessary. Prepare three to five master stocks of each clone for long-term storage. After receiving the bacterial stab cultures of Gateway Full ORF Clones, you should:

1. Streak a small portion of the bacterial stab culture on a LB plate containing 50 µg/ml kanamycin. Incubate the plate at 37°C overnight.

2. Isolate a single colony and inoculate 5–10 ml of LB containing 50 µg/ml kanamycin. 3. Grow the culture to stationary phase (OD600 = 1–2). 4. Mix 0.8 ml of culture with 0.2 ml of sterile glycerol and transfer to a kryovial. 5. Store at -80°C. Use one master stock to create working stocks for regular use.



5.2. Plasmid preparation by alkaline lysis

Plasmids of Gateway Full ORF Clones can be isolated using standard plasmid isolation techniques, which are in use in your lab. Alkaline Lysis procedure according to Sambrook et al., 1989, with modifications.

1. Grow clone in 2 ml LB/kan (50 µg/ml) for 18-20 h with strong agitation (250 rpm) 2. Spin down 1.5 ml bacterial culture at >12000 xg for 30 sec at 4°C. 3. Remove the supernatant leaving the pellet as dry as possible, i.e. decant and then

remove traces of medium with a pipette or use a vacuum line. 4. Resuspend the pellet in 100 µl ice-cold AlkLysI by vigorous vortexing. 5. Add 200 µl freshly prepared AlkLysII, mix by inverting 5 times and store on ice for 15 min. 6. Add 150 µl AlkLysIIIB, mix by inverting the tube 5 times, store on ice for at least 3 min. 7. Spin > 12000 xg, 4°C, 5 min

• (Optional, but strongly recommended): add 450 µl phenol/chloroform to the supernatant and extract, spin at >12000 xg for 2 min.

8. Precipitate the DNA by addition of 900 µl EtOH, mix by vortexing, and let sit at room temperature for at least 2 min.

9. Spin > 12000 xg at 4°C for 5 min and remove the supernatant carefully. 10. Wash pellet with 1 ml 70% EtOH at 4°C. 11. Spin as in 9. 12. Dry pellet at room temperature for 10 min. 13. Resuspend in 50 µl TE. 14. Check 1 µl on a gel, either digested or undigested. 15. The average yield is about 3 µg/1.5 ml culture, when Gateway Full ORF Clones are used.

This is approx. 60 ng/µl. AlkLys Solution I: 25 mM Tris/Cl (pH 8,0) 25 ml (1 M) 10 mM EDTA (pH 8,0 or 7,6) 20 ml (0.5 M) 50 mM α-D-Glucose 9,1 g H2O add 1 l AlkLys Solution II: 0,2 M NaOH 1% SDS Prepare fresh every time. AlkLys Solution III B: 3 M KAc 60 ml (5 M) or 29,45 g Glacial acetic acid 11,5 ml 11,5 ml H2O 28,5 ml to 100 ml Phenol/chloroform/isoamylalcohol (25:24:1) Mix equal volumes (1 l) of CHCl3 and melted phenol and add 1 M Tris base and mix vigorously. Let settle and check pH. Repeat this procedure until pH of aqueous phase is about 8.0. Remove aqueous phase and extract twice with 100 ml TE (pH 8.0). Aliquot and store in dark bottle at 4°C. TE (pH 8,0), 10:1 10 mM Tris/Cl (pH 8,0) 1 mM EDTA Add 10 ml 1 M Tris/Cl (pH 8,0) and 2 ml 0,5 M EDTA (pH 8,0) to 900 ml sterile MQ water, check pH, adjust volume to 1 l and dispense into aliquots.



5.3. Other plasmid preparation methods

Many biochemical suppliers support plasmid minipreparations with appropriate kits, e.g. Qiagen, Roche, Machery & Nagel, Clontech, Invitrogen, Stratagene, GE and others. 5.4. Restriction digest of Gateway Full ORF Clone plasmids

In order to characterize the insert size of a Gateway Full ORF Clone, we recommend cutting the plasmids to be tested using the restriction enzyme BsrGI (e.g. obtained from New England Biolabs). BsrGI restriction site (T|GTACA) is present in attL sides, both attL1 and attL2 that flank the insert of all Gateway Full ORF Clones. Therefore a BsrGI cut will release the insert, but won’t cut in the backbone of the vectors (pDONR201 and pDONR221, respectively).

1. Mix on ice: 2. 200–400 ng plasmid DNA

1 µl 10x restriction buffer, supplied with the enzyme 1 U BsrGI restriction enzyme to 10 µl water

3. Incubate at 37°C for 2h 4. Analyze the digest on a 1 % agarose gel using appropriate size markers (range 200 bp to

at least 4 kb) Expect a vector fragment of 2,2 kb for pDONR201 (or 2,5 kb for pDONR221), and an insert band whose size depends on the size of the ORF. In some cases there will be multiple bands beside the vector fragment. This can be observed whenever the ORF sequence contains one or more BsrGI recognition sites. Please verify the presence of BsrGI sites in the insert by checking the ORF sequence.



5.5. Shuttling full ORFs by Gateway® LR reaction

We have optimized different types of LR Gateway® reaction protocols to fulfill different criteria. Please apply the fast protocol if quick access to destination clones is required. This protocol can also be automated easily. The high-confidence protocol ensures the most reliable shuttling of an ORF of interest into the destination vector you are using, by maximizing the reliability of the LR reaction and the amount of DNA and enzyme. Finally, the multi-destination protocol is most convenient and cost-saving whenever you wish to shuttle one insert into multiple destination vectors for multiple applications at the same time. Always use Invitrogens LR Clonase to perform the Gateway® LR reaction. 5.5.1. Fast Gateway® LR protocol

This protocol allows you to create Full ORF Expression Clones within 1 day. You will perform the LR recombination reaction to transfer the ORF into a destination vector of your choice (attR1/2-cassette-containing vector), to create an attB-containing expression clone.

1. For a single BP reaction, assemble on ice: 2 µl 5x LR buffer Gateway® 3 µl destination vector (150 ng/µl) 3 µl water 0,5 µl Topoisomerase I (alternatively use linearized destination vector; linearize using restriction enzyme cutting between ccdB and CmR genes of the Gateway® cassette) 0,5 µl LR Clonase total 10 µl

2. Incubate for 2 h at 25°C (preferably in PCR machine with heated lid or in water bath with cover). O/n incubation results in an at least 5x higher efficiency.

3. Use immediately 5 µl for chemical transformation into standard E. coli (transformation protocol see above, or use your favorite protocol).

4. Plate the transformation onto LB/antibiotic plate (using the antibiotic that is coded by your destination vector).

5. Incubate o/n at 37°C 6. Pick at least two colonies and characterize the insert (see notes)

Note:

a. LR reaction efficiency is usually at > 90–95%, so the vast majority of the clones contain exactly the right ORF in the frame that is defined by the Gateway Full ORF Clone and the destination vector used. Therefore many users pick a single colony and use this clone without further verification.



5.5.2. High Confidence Gateway® LR protocol

This protocol allows you to create Full ORF Expression Clones with high confidence within 3 days.

1. For a single BP reaction, assemble on ice: 4 µl 5x LR buffer 6 µl destination vector (200–250 ng/µl) 2 µl Gateway Full ORF Clone (150–250 ng/µl, or 1 µl of a miniprep) 6 µl water 1 µl Topoisomerase I (alternatively use linearized destination vector; linearize using restriction enzyme cutting between ccdB and CmR genes of the Gateway® cassette) 1 µl LR Clonase total 20 µl

2. Incubate o/n at 25°C (preferably in PCR machine with heated lid or in water bath with cover, see notes).

3. Add 2 µl stop mix (2 µg/µl Proteinase K), and incubate for 10 min at 37°C. 4. Add:

2 µl glycogen / Pellet Paint 4 µl 3M Na Acetate pH 5.2 100 µl EtOH abs., and mix

5. Spin at 13.000 rpm for 15 min. 6. Wash pellet with at least 200 µl 70% EtOH. 7. Resuspend dried pellet in 5 µl water. 8. Use 1 µl for transformation of electrocompetent E. coli that have a transformation

efficiency of 109 cfu/µg plasmid. We recommend T-phage-resistant bacteria. 9. Plate the transformation onto LB/antibiotic plate (using the antibiotic that is coded by your

destination vector). 10. Incubate o/n at 37°C 11. Pick at least two colonies and characterize the insert (see notes)

Notes:

a. Prolonged incubation of LR reaction increases the transformation efficiency significantly. This is especially important when shuttling long inserts (> 2,5 kb)

b. LR reaction efficiency is usually at > 90–95%, so the vast majority of the clones contain exactly the right ORF in the frame that is defined by the Gateway Full ORF Clone and the destination vector used. Therefore many users pick a single colony and use this clone without further verification.



5.5.3. Multi Destination Gateway® LR protocol

This protocol allows you to create multiple different Full ORF Expression Clones (vector constructs) of a single ORF via one Gateway® LR reaction and transformation. Make sure that these destination vectors have different antibiotic resistance genes. Avoid kanamycin and chloramphenicol, as these resistances are coded by the Gateway Full ORF Clone, and the Gateway® cassette that are present in the destination vectors, respectively.

1. For a multi-destination BP reaction, assemble on ice: 4 µl 5x LR buffer Gateway® 1 µl destination vector 1 (100–250 ng/µl) - see notes 1 µl destination vector 2 (100–250 ng/µl) optional 1 µl destination vector 3 (100–250 ng/µl) optional 1 µl destination vector 4 (100–250 ng/µl) 2 µl Gateway Full ORF Clone (150–250 ng/µl) to 18 µl water 1 µl Topoisomerase I (alternatively use linearized destination vectors; linearize using restriction enzyme cutting between ccdB and CmR genes of the Gateway® cassette) 1 µl LR Clonase total 20 µl

2. Incubate o/n at 25°C (preferably in PCR machine with heated lid or in water bath with cover, see notes).

3. Add 2 µl stop mix (2 µg/µl Proteinase K), and incubate for 10 min at 37°C. 4. Add: 2 µl Glycogen / Pellet Paint

4 µl 3M Na Acetate pH 5.2 100 µl EtOH abs., and mix

5. Spin at 13.000 rpm for 15 min. 6. Wash pellet with at least 200 µl 70% EtOH. 7. Resuspend dried pellet in 5 µl water. 8. Use 1 µl for transformation of electrocompetent E. coli that have a transformation

efficiency of 109 cfu/µg plasmid. We recommend T-phage-resistant bacteria. 9. Plate equal amounts of the transformation onto LB/antibiotic1, LB/antibiotic2,

(LB/antibiotic3, LB/antibiotic4) plates (using the antibiotics that are coded by your respective destination vectors, see notes).

10. Incubate o/n at 37°C 11. Pick at least two colonies and characterize the insert (see notes)

Notes: a. Up to four different destination vectors have been used successfully in a single LR

reaction. Reduce the amount of each single vector accordingly to give 1 µg total destination vector mix per LR reaction. The destination vectors used in combination MUST code for different antibiotic resistances. Kanamycin and chloramphenicol should NOT be amongst the antibiotics used as resistance genes to these are already carried on the Gateway Full ORF Clones and the Gateway® cassette, respectively.

b. Prolonged incubation of LR reaction increases the transformation efficiency significantly. This is especially important when shuttling long inserts (> 2,5 kb).

c. In order to achieve single colonies, we recommended that plating should be done at two concentrations on two identical LB/antibiotics plates (e.g. 20 µl and 200 µl aliquots).

d. LR reaction efficiency is usually at > 90–95%, so the vast majority of the clones contain exactly the right ORF in the frame that is defined by the Gateway Full ORF Clone and the destination vector used. Therefore many users pick a single colony and use this clone without further verification.



5.6. Preparation of chemically competent E. coli

1. Inoculate 1 ml o/n-culture of phage-resistant E. coli (e.g. DH5α T phage-resistant) in 100 ml LB; incubate on shaker at 37°C.

2. Harvest in log phase at OD600 = 0,3–0,4, by spinning at 3600 rpm at 4°C for 10 min. 3. Resuspend in 50 ml 100 mM CaCl2 (ice-cold). 4. Leave on ice for 30 min and spin down as above.

a. For immediate use: Resuspend in 5 ml 100 mM CaCl2 (ice-cold). Storage at 4°C o/n increases transformation efficiency approximately five-fold.

b. For long-term storage: Resuspend in < 5 ml 100 mM CaCl2, 15% glycerol (ice-cold).Freeze in 200 µl aliquots on dry ice and store at -80°C.

Notes: a. E. coli prepared according to this protocol usually have a transformation efficiency of at

least 106/µg supercoiled plasmid. b. It is absolutely essential that all steps of this protocol are performed as cold as possible,

this means: never remove cells from ice once they have been harvested. LB (Luria-Bertani) medium 10 g bacto-tryptone 5 g bacto-yeast extract 10 g NaCl Dissolve in 950 ml VE water, and adjust pH to 7 with 5 N NaOH (about 200 µl), if necessary. Fill up to 1 l and autoclave. 5.7. Transformation of Gateway® LR reactions

1. Thaw on ice (takes about 15 min). 2. Transfer 100 µl aliquots to pre-cooled and labeled 15 ml Falcon tubes. 3. Add 1,7 µl ß-mercaptoethanol (1,44 M) to achieve a final concentration of 25 mM. Mix

cells and ß-mercaptoethanol by swirling the tube. 4. Incubate on ice for 10 min. 5. Add 1–3 µl of a Gateway® LR Clonase reaction to the bacteria. 6. Leave on ice for 30 min. 7. Heat shock at 42°C for 30 sec. 8. Add 0,9 ml of LB medium and incubate at 37°C on shaker for 30–60 min, 250 rpm. 9. Plate 25 µl and 250 µl of the transformation onto LB/antibiotic (according to the resistance

gene(s) carried by the destination/expression plasmids) plates. Store the remaining transformation at 4°C.

10. Incubate the plate at 37°C overnight. 11. Isolate a single colony and inoculate into 5–10 ml of LB containing appropriate antibiotic. 12. Grow the culture to stationary phase (OD600=1–2). 13. Mix 0.8 ml of culture with 0.2 ml of sterile glycerol and transfer to a kryovial. 14. Store at -80°C. Use one master stock to create working stocks for regular use.



6. Appendix

6.1. Gateway Full ORF Clones: Vector Information

6.1.1. pDONR201 features

Description of the features in the vicinity of the cloning site of pDONR201. Please verify the nature of the cloning vector (pDONR201 or pDONR221). ------------------>

tttatttgat gcctggcagt tccctactct cgcgttaacg ctagcatgga tctcgggccc

don5

caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa

attL1 tgctttttta taatgccaag tttgtacaaa aaa gca ggc –stuffer– ATG-ORF –stuffer-

ac cca gct ttc ttgtacaaa gtgggcatta taagaaagca ttgcttatca atttgttgca

attL2

acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct gcagctctgg

<----------------------

cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc

don3 red: attL1/2 gray: Stuffer between attL sites and ORF. This stuffer may vary in different Gateway Full ORF

Clones, please consult the GPCF web page and check for der exact clone sequence. bold: ORF sequence blue: Sequencing and PCR primers don5 and don3 yyyyyy Sequence underlined in green corresponds to the sequence fragment transferred to

destinations clones by LR reaction.



6.1.2. pDONR201 map

black bar: origin of replication orange bars: coding sequences (kanamycin resistance and ORF of interest) blue bars: terminator sequences red letters: primer binding sites (sequences see “Oligonucleotide” section) black letters: restriction enzyme sites (single cutter in vector) gray letters: restriction enzyme sites (dual cutter in vector)

pDONR201 Shuttle Clone

Kan(R)

dummy-ORF

pUC ori

rrnB T1 transcription terminator


AcyI (133)AflIII (3098)

ApaI (330)

ApaLI (2784)

Bsp120I (326)

HpaI (307)

PstI (1392)PvuII (1387)

BsrGI (414)

BsrGI (1292)

NheI (45)

NheI (311)

don3 (100.0%)

don5 (100.0%)

pDONR201 Gateway Entry Vector

(with dummy insert)




Description of the features in the vicinity of the cloning site of pDONR201. Please verify the nature of the cloning vector (pDONR201 or pDONR221). ------------------>

tttatttgat gcctggcagt tccctactct cgcgttaacg ctagcatgga tgttttccca

don5 ---------------------->

gtcacgacgt tgtaaaacga cggccagtct taagctcggg ccccaaataa tgattttatt

M13u

ttgactgata gtgacctgtt cgttgcaaca cattgatgag caatgctttt ttataatgcc

attL1

aactttgtac aaa aaa gca ggc –stuffer– ATG-ORF –stuffer– ac cca gct

ttc ttg tacaaa gttggcatta taagaaagca ttgcttatca atttgttgca

attL2

acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct gatatcccct

<------------------

atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa

M13r <----------------------

atctctgatg ttacattgca caagataaaa taatatc

don3

red: attL1/2 gray: Stuffer between attL sites and ORF. This stuffer may vary in different Gateway Full ORF

Clones, please consult the GPCF web page and check for der exact clone sequence. bold: ORF sequence blue: Sequencing and PCR primers don5, don3, M13u, M13r yyyyyy Sequence underlined in green corresponds to the sequence fragment transferred to

destinations clones by LR reaction.



6.1.4. pDONR221 map

black bars: origin of replication orange bars: coding sequences (kanamycin resistance and ORF of interest) blue bars: terminator sequences green bars attL recombination sites (used in LR Gateway® reaction) red letters: primer binding sites black letters: restriction enzyme sites (single cutter in vector) gray letters: restriction enzyme sites (dual cutter in vector)

pDONR221 Shuttle Clone

Kan(R)dummy-ORF

pUC orirrnB T1 transcription terminator


attL1

attL2

Acy I (328)

Apa I (568)

Apa LI (3071)

Asp EI (550)Bsp 120I (564)

Eco RV (1628)

Hpa I (502)

Bsr GI (652)

Bsr GI (1528)

Mlu I (231)

Mlu I (2689)

Nhe I (240)

Nhe I (506)

Pvu II (175)

Pvu II (1623)

M13u (100.0%)

don3 (100.0%)

don5 (100.0%)

M13r (100.0%)

pDONR221 Gateway Entry Vector

(with dummy insert)




See sequence of pDONR223 – chapter 6.3.2 below Important: pDONR223 carries the Spetinomycin selection marker for clone propagation in E.coli. 6.1.6. pDONR223 map



6.2. Genotypes of E. coli

Gateway destination vectors are delivered in DB3.1 strains, suppressing ccdB function. Gateway Full ORF Clones are provided in DH5 or DH10 strains.

DH5α: F- φ80dlacZ∆M15∆(lacZYA-argF) U169 endA1 recA1 hsdR17(rK- mK+) deoR thi-1 supE44 λ- gyrA96 relA1 FOCUS (1986) 8:2, 9

DH5α T1-resistant: F- φ80dlacZ∆M15∆(lacZYA-argF) U169 endA1 recA1 hsdR17(rK- mK+) deoR thi-1 supE44 λ- gyrA96 relA1 tonA

DH10B: F- mcrA ∆(mrr-hsdRMS-mcrBC) φ80dlacZ∆M15 ∆lacX74 endA1 recA1 deoR ∆(ara, leu)7697 araD139 galU galK nupG rpsL λ- Lorow, D. and Jessee, J. (1990) FOCUS 12, 19

DH10B T-phage-resistant: F- mcrA ∆(mrr-hsdRMS-mcrBC) φ80dlacZ∆M15 ∆lacX74 endA1 recA1 deoR ∆(ara, leu)7697 araD139 galU galK nupG rpsL λ- tonA

DB3.1: F- gyrA462 endA1 glnV44 Δ(sr1-recA) mcrB mrr hsdS20(rB-, mB

-) ara14 galK2 lacY1 proA2 rpsL20(Smr) xyl5 Δleu mtl1

6.3. Sequences

6.3.1. Primers

The Gateway Full ORF Clones may be characterized by PCR and/or sequencing. Please use the recommended oligonucleotides sequences provided below.

don5: cgttaacgctagcatgga don3: tcttgtgcaatgtaacatcag M13u: cgttgtaaaacgacggccagt M13r: ccaggaaacagctatgac



6.3.2. Vectors

> pDONR201 1 gttaacgcta gcatggatct cgggccccaa ataatgattt tattttgact gatagtgacc

61 tgttcgttgc aacaaattga tgagcaatgc ttttttataa tgccaacttt gtacaaaaaa

121 gctgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat tttgcataaa

181 aaacagacta cataatactg taaaacacaa catatccagt cactatgaat caactactta

241 gatggtatta gtgacctgta gtcgaccgac agccttccaa atgttcttcg ggtgatgctg

301 ccaacttagt cgaccgacag ccttccaaat gttcttctca aacggaatcg tcgtatccag

361 cctactcgct attgtcctca atgccgtatt aaatcataaa aagaaataag aaaaagaggt

421 gcgagcctct tttttgtgtg acaaaataaa aacatctacc tattcatata cgctagtgtc

481 atagtcctga aaatcatctg catcaagaac aatttcacaa ctcttatact tttctcttac

541 aagtcgttcg gcttcatctg gattttcagc ctctatactt actaaacgtg ataaagtttc

601 tgtaatttct actgtatcga cctgcagact ggctgtgtat aagggagcct gacatttata

661 ttccccagaa catcaggtta atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag

721 ccacttcttc cccgataacg gagaccggca cactggccat atcggtggtc atcatgcgcc

781 agctttcatc cccgatatgc accaccgggt aaagttcacg ggagacttta tctgacagca

841 gacgtgcact ggccaggggg atcaccatcc gtcgcccggg cgtgtcaata atatcactct

901 gtacatccac aaacagacga taacggctct ctcttttata ggtgtaaacc ttaaactgca

961 tttcaccagt ccctgttctc gtcagcaaaa gagccgttca tttcaataaa ccgggcgacc

1021 tcagccatcc cttcctgatt ttccgctttc cagcgttcgg cacgcagacg acgggcttca

1081 ttctgcatgg ttgtgcttac cagaccggag atattgacat catatatgcc ttgagcaact

1141 gatagctgtc gctgtcaact gtcactgtaa tacgctgctt catagcacac ctctttttga

1201 catacttcgg gtatacatat cagtatatat tcttataccg caaaaatcag cgcgcaaata

1261 cgcatactgt tatctggctt ttagtaagcc ggatccacgc gattacgccc cgccctgcca

1321 ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc catcacagac

1381 ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt

1441 gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact

1501 ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg

1561 gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg

1621 ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa

1681 aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat

1741 acggaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa

1801 cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg

1861 gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg

1921 ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc

1981 tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa

2041 gttggaacct cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg cccagggctt

2101 cccggtatca acagggacac caggatttat ttattctgcg aagtgatctt ccgtcacagg

2161 tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac ttagtcgact acaggtcact

2221 aataccatct aagtagttga ttcatagtga ctggatatgt tgtgttttac agtattatgt

2281 agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc

2341 tcgttcagct ttcttgtaca aagttggcat tataagaaag cattgcttat caatttgttg

2401 caacgaacag gtcactatca gtcaaaataa aatcattatt tgccatccag ctgcagctct

2461 ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa



2521 caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac

2581 gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat

2641 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg

2701 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg

2761 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta

2821 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc

2881 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc

2941 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc

3001 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg

3061 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat

3121 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg

3181 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg

3241 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt

3301 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg

3361 atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga

3421 cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg

3481 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt

3541 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca

3601 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac

3661 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac

3721 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct

3781 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg

3841 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca

3901 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt

3961 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta

4021 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc

4081 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc

4141 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa

4201 ccgtattacc gctagccagg aagagtttgt agaaacgcaa aaaggccatc cgtcaggatg

4261 gccttctgct tagtttgatg cctggcagtt tatggcgggc gtcctgcccg ccaccctccg

4321 ggccgttgct tcacaacgtt caaatccgct cccggcggat ttgtcctact caggagagcg

4381 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt ccgactgagc ctttcgtttt

4441 atttgatgcc tggcagttcc ctactctcgc



> pDONR221 1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga

61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga

121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca

181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc

241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta

301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc

361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa

421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg

481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa

541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac

601 ctgttcgttg caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa

661 agctgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa

721 aaaacagact acataatact gtaaaacaca acatatccag tcactatgaa tcaactactt

781 agatggtatt agtgacctgt agtcgaccga cagccttcca aatgttcttc gggtgatgct

841 gccaacttag tcgaccgaca gccttccaaa tgttcttctc aaacggaatc gtcgtatcca

901 gcctactcgc tattgtcctc aatgccgtat taaatcataa aaagaaataa gaaaaagagg

961 tgcgagcctc ttttttgtgt gacaaaataa aaacatctac ctattcatat acgctagtgt

1021 catagtcctg aaaatcatct gcatcaagaa caatttcaca actcttatac ttttctctta

1081 caagtcgttc ggcttcatct ggattttcag cctctatact tactaaacgt gataaagttt

1141 ctgtaatttc tactgtatcg acctgcagac tggctgtgta taagggagcc tgacatttat

1201 attccccaga acatcaggtt aatggcgttt ttgatgtcat tttcgcggtg gctgagatca

1261 gccacttctt ccccgataac ggagaccggc acactggcca tatcggtggt catcatgcgc

1321 cagctttcat ccccgatatg caccaccggg taaagttcac gggagacttt atctgacagc

1381 agacgtgcac tggccagggg gatcaccatc cgtcgcccgg gcgtgtcaat aatatcactc

1441 tgtacatcca caaacagacg ataacggctc tctcttttat aggtgtaaac cttaaactgc

1501 atttcaccag cccctgttct cgtcagcaaa agagccgttc atttcaataa accgggcgac

1561 ctcagccatc ccttcctgat tttccgcttt ccagcgttcg gcacgcagac gacgggcttc

1621 attctgcatg gttgtgctta ccagaccgga gatattgaca tcatatatgc cttgagcaac

1681 tgatagctgt cgctgtcaac tgtcactgta atacgctgct tcatagcata cctctttttg

1741 acatacttcg ggtatacata tcagtatata ttcttatacc gcaaaaatca gcgcgcaaat

1801 acgcatactg ttatctggct tttagtaagc cggatccacg cggcgtttac gccccgccct

1861 gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac

1921 agacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat

1981 atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa

2041 aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt

2101 tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa

2161 actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat

2221 ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg

2281 ccatacggaa ttccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat

2341 aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg

2401 tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc

2461 attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag

2521 ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt

2581 gaaagttgga acctcttacg tgccgatcaa cgtctcattt tcgccaaaag ttggcccagg

2641 gcttcccggt atcaacaggg acaccaggat ttatttattc tgcgaagtga tcttccgtca

2701 caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc caacttagtc gactacaggt

2761 cactaatacc atctaagtag ttgattcata gtgactggat atgttgtgtt ttacagtatt

2821 atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg

2881 tttctcgttc agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt

2941 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata



3001 tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt

3061 ctcaaaatct ctgatgttac attgcacaag ataaaataat atcatcatga acaataaaac

3121 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt

3181 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg

3241 ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag

3301 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca

3361 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc

3421 ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc caggtattag

3481 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt

3541 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc

3601 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta

3661 atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg

3721 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat

3781 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca

3841 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat

3901 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt

3961 tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg acttgacggg

4021 acggcgcaag ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt

4081 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct

4141 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc

4201 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc

4261 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc

4321 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg

4381 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt

4441 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg

4501 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg

4561 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt

4621 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag

4681 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt

4741 gctggccttt tgctcacatg tt

>pDONR223 LOCUS pDONR223 5005 bp DNA SYN DEFINITION pDONR223 ACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors. FEATURES Location/Qualifiers source 1..5005 /organism="pDONR223" /mol_type="other DNA" terminator complement(268..295) /label="rrnB_T2_terminator" terminator complement(427..470) /label="rrnB_T1_terminator" misc_feature 522..544 /label="M13_pUC_fwd_primer" promoter 537..553 /label="M13_forward20_primer" misc_feature complement(569..652) /label="attL1" misc_feature 570..801 /label="attP" misc_feature 656..768 /label="attR1"



misc_feature complement(668..768) /label="attR2" misc_feature complement(1197..1502) /label="ccdB" gene complement(1844..2503) /label="CAT/CamR" /gene="CAT/CamR" CDS complement(1844..2503) /label="ORF frame 1" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF LKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETF SSLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNV ANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG A*" misc_feature complement(2751..2982) /label="attP" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF LKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETF SSLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNV ANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG A*" misc_feature complement(2784..2891) /label="attR1" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF LKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETF SSLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNV ANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG A*" misc_feature 2784..2884 /label="attR2" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF LKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETF SSLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNV ANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG A*" misc_feature 2896..2983 /label="attL2" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF LKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETF SSLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNV ANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG A*" promoter complement(3001..3019)



/label="T7_promoter" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF LKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETF SSLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNV ANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG A*" promoter complement(3020..3038) /label="M13_reverse_primer" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF LKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETF SSLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNV ANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG A*" gene 3252..4262 /label="spect" /gene="spect" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF LKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETF SSLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNV ANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG A*" CDS 3252..4262 /label="ORF frame 3" /translation="MRSRNWSRTLTERSGGNGAVAVFMACYDCFFGVQSMPRASKQQA RYAVGRCLMLWSSNDVTQQGSRPKTKLNIMREAVIAEVSTQLSEVVGVIERHLEPTLL AVHLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVE VTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVAL VGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIA PKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVV GK*" rep_origin 4339..4958 /label="pBR322_origin" /translation="MRSRNWSRTLTERSGGNGAVAVFMACYDCFFGVQSMPRASKQQA RYAVGRCLMLWSSNDVTQQGSRPKTKLNIMREAVIAEVSTQLSEVVGVIERHLEPTLL AVHLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVE VTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVAL VGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIA PKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVV GK*"



ORIGIN 1 CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCTTTG AGTGAGCTGA 61 TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG AAGCGGAAGA 121 GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 181 CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATA CGCGTACCGC 241 TAGCCAGGAA GAGTTTGTAG AAACGCAAAA AGGCCATCCG TCAGGATGGC CTTCTGCTTA 301 GTTTGATGCC TGGCAGTTTA TGGCGGGCGT CCTGCCCGCC ACCCTCCGGG CCGTTGCTTC 361 ACAACGTTCA AATCCGCTCC CGGCGGATTT GTCCTACTCA GGAGAGCGTT CACCGACAAA 421 CAACAGATAA AACGAAAGGC CCAGTCTTCC GACTGAGCCT TTCGTTTTAT TTGATGCCTG 481 GCAGTTCCCT ACTCTCGCGT TAACGCTAGC ATGGATGTTT TCCCAGTCAC GACGTTGTAA 541 AACGACGGCC AGTCTTAAGC TCGGGCCCCA AATAATGATT TTATTTTGAC TGATAGTGAC 601 CTGTTCGTTG CAACAAATTG ATGAGCAATG CTTTTTTATA ATGCCAACTT TGTACAAAAA 661 AGCTGAACGA GAAACGTAAA ATGATATAAA TATCAATATA TTAAATTAGA TTTTGCATAA 721 AAAACAGACT ACATAATACT GTAAAACACA ACATATCCAG TCACTATGAA TCAACTACTT 781 AGATGGTATT AGTGACCTGT AGTCGACCGA CAGCCTTCCA AATGTTCTTC GGGTGATGCT 841 GCCAACTTAG TCGACCGACA GCCTTCCAAA TGTTCTTCTC AAACGGAATC GTCGTATCCA 901 GCCTACTCGC TATTGTCCTC AATGCCGTAT TAAATCATAA AAAGAAATAA GAAAAAGAGG 961 TGCGAGCCTC TTTTTTGTGT GACAAAATAA AAACATCTAC CTATTCATAT ACGCTAGTGT 1021 CATAGTCCTG AAAATCATCT GCATCAAGAA CAATTTCACA ACTCTTATAC TTTTCTCTTA 1081 CAAGTCGTTC GGCTTCATCT GGATTTTCAG CCTCTATACT TACTAAACGT GATAAAGTTT 1141 CTGTAATTTC TACTGTATCG ACCTGCAGAC TGGCTGTGTA TAAGGGAGCC TGACATTTAT 1201 ATTCCCCAGA ACATCAGGTT AATGGCGTTT TTGATGTCAT TTTCGCGGTG GCTGAGATCA 1261 GCCACTTCTT CCCCGATAAC GGAGACCGGC ACACTGGCCA TATCGGTGGT CATCATGCGC 1321 CAGCTTTCAT CCCCGATATG CACCACCGGG TAAAGTTCAC GGGAGACTTT ATCTGACAGC 1381 AGACGTGCAC TGGCCAGGGG GATCACCATC CGTCGCCCGG GCGTGTCAAT AATATCACTC 1441 TGTACATCCA CAAACAGACG ATAACGGCTC TCTCTTTTAT AGGTGTAAAC CTTAAACTGC 1501 ATTTCACCAG TCCCTGTTCT CGTCAGCAAA AGAGCCGTTC ATTTCAATAA ACCGGGCGAC 1561 CTCAGCCATC CCTTCCTGAT TTTCCGCTTT CCAGCGTTCG GCACGCAGAC GACGGGCTTC 1621 ATTCTGCATG GTTGTGCTTA CCAGACCGGA GATATTGACA TCATATATGC CTTGAGCAAC 1681 TGATAGCTGT CGCTGTCAAC TGTCACTGTA ATACGCTGCT TCATAGCACA CCTCTTTTTG 1741 ACATACTTCG GGTATACATA TCAGTATATA TTCTTATACC GCAAAAATCA GCGCGCAAAT 1801 ACGCATACTG TTATCTGGCT TTTAGTAAGC CGGATCCACG CGATTACGCC CCGCCCTGCC 1861 ACTCATCGCA GTACTGTTGT AATTCATTAA GCATTCTGCC GACATGGAAG CCATCACAGA 1921 CGGCATGATG AACCTGAATC GCCAGCGGCA TCAGCACCTT GTCGCCTTGC GTATAATATT 1981 TGCCCATGGT GAAAACGGGG GCGAAGAAGT TGTCCATATT GGCCACGTTT AAATCAAAAC 2041 TGGTGAAACT CACCCAGGGA TTGGCTGAGA CGAAAAACAT ATTCTCAATA AACCCTTTAG 2101 GGAAATAGGC CAGGTTTTCA CCGTAACACG CCACATCTTG CGAATATATG TGTAGAAACT 2161 GCCGGAAATC GTCGTGGTAT TCACTCCAGA GCGATGAAAA CGTTTCAGTT TGCTCATGGA 2221 AAACGGTGTA ACAAGGGTGA ACACTATCCC ATATCACCAG CTCACCGTCT TTCATTGCCA 2281 TACGGAATTC CGGATGAGCA TTCATCAGGC GGGCAAGAAT GTGAATAAAG GCCGGATAAA 2341 ACTTGTGCTT ATTTTTCTTT ACGGTCTTTA AAAAGGCCGT AATATCCAGC TGAACGGTCT 2401 GGTTATAGGT ACATTGAGCA ACTGACTGAA ATGCCTCAAA ATGTTCTTTA CGATGCCATT 2461 GGGATATATC AACGGTGGTA TATCCAGTGA TTTTTTTCTC CATTTTAGCT TCCTTAGCTC 2521 CTGAAAATCT CGATAACTCA AAAAATACGC CCGGTAGTGA TCTTATTTCA TTATGGTGAA 2581 AGTTGGAACC TCTTACGTGC CGATCAACGT CTCATTTTCG CCAAAAGTTG GCCCAGGGCT 2641 TCCCGGTATC AACAGGGACA CCAGGATTTA TTTATTCTGC GAAGTGATCT TCCGTCACAG 2701 GTATTTATTC GGCGCAAAGT GCGTCGGGTG ATGCTGCCAA CTTAGTCGAC TACAGGTCAC 2761 TAATACCATC TAAGTAGTTG ATTCATAGTG ACTGGATATG TTGTGTTTTA CAGTATTATG 2821 TAGTCTGTTT TTTATGCAAA ATCTAATTTA ATATATTGAT ATTTATATCA TTTTACGTTT 2881 CTCGTTCAGC TTTCTTGTAC AAAGTTGGCA TTATAAGAAA GCATTGCTTA TCAATTTGTT 2941 GCAACGAACA GGTCACTATC AGTCAAAATA AAATCATTAT TTGCCATCCA GCTGATATCC 3001 CCTATAGTGA GTCGTATTAC ATGGTCATAG CTGTTTCCTG GCAGCTCTGG CCCGTGTCTC 3061 AAAATCTCTG ATGTTACATT GCACAAGATA AAAATATATC ATCATGCCTC CTCTAGACCA 3121 GCCAGGACAG AAATGCCTCG ACTTCGCTGC TGCCCAAGGT TGCCGGGTGA CGCACACCGT 3181 GGAAACGGAT GAAGGCACGA ACCCAGTGGA CATAAGCCTG TTCGGTTCGT AAGCTGTAAT 3241 GCAAGTAGCG TATGCGCTCA CGCAACTGGT CCAGAACCTT GACCGAACGC AGCGGTGGTA 3301 ACGGCGCAGT GGCGGTTTTC ATGGCTTGTT ATGACTGTTT TTTTGGGGTA CAGTCTATGC 3361 CTCGGGCATC CAAGCAGCAA GCGCGTTACG CCGTGGGTCG ATGTTTGATG TTATGGAGCA 3421 GCAACGATGT TACGCAGCAG GGCAGTCGCC CTAAAACAAA GTTAAACATC ATGAGGGAAG 3481 CGGTGATCGC CGAAGTATCG ACTCAACTAT CAGAGGTAGT TGGCGTCATC GAGCGCCATC 3541 TCGAACCGAC GTTGCTGGCC GTACATTTGT ACGGCTCCGC AGTGGATGGC GGCCTGAAGC 3601 CACACAGTGA TATTGATTTG CTGGTTACGG TGACCGTAAG GCTTGATGAA ACAACGCGGC 3661 GAGCTTTGAT CAACGACCTT TTGGAAACTT CGGCTTCCCC TGGAGAGAGC GAGATTCTCC



3721 GCGCTGTAGA AGTCACCATT GTTGTGCACG ACGACATCAT TCCGTGGCGT TATCCAGCTA 3781 AGCGCGAACT GCAATTTGGA GAATGGCAGC GCAATGACAT TCTTGCAGGT ATCTTCGAGC 3841 CAGCCACGAT CGACATTGAT CTGGCTATCT TGCTGACAAA AGCAAGAGAA CATAGCGTTG 3901 CCTTGGTAGG TCCAGCGGCG GAGGAACTCT TTGATCCGGT TCCTGAACAG GATCTATTTG 3961 AGGCGCTAAA TGAAACCTTA ACGCTATGGA ACTCGCCGCC CGACTGGGCT GGCGATGAGC 4021 GAAATGTAGT GCTTACGTTG TCCCGCATTT GGTACAGCGC AGTAACCGGC AAAATCGCGC 4081 CGAAGGATGT CGCTGCCGAC TGGGCAATGG AGCGCCTGCC GGCCCAGTAT CAGCCCGTCA 4141 TACTTGAAGC TAGACAGGCT TATCTTGGAC AAGAAGAAGA TCGCTTGGCC TCGCGCGCAG 4201 ATCAGTTGGA AGAATTTGTC CACTACGTGA AAGGCGAGAT CACCAAGGTA GTCGGCAAAT 4261 AACCCTCGAG CCACCCATGA CCAAAATCCC TTAACGTGAG TTACGCGTCG TTCCACTGAG 4321 CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT CTGCGCGTAA 4381 TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT GGTTTGTTTG CCGGATCAAG 4441 AGCTACCAAC TCTTTTTCCG AAGGTAACTG GCTTCAGCAG AGCGCAGATA CCAAATACTG 4501 TCCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA CCGCCTACAT 4561 ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG TCGTGTCTTA 4621 CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC TGAACGGGGG 4681 GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA TACCTACAGC 4741 GTGAGCATTG AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG TATCCGGTAA 4801 GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC AGGGGGAAAC GCCTGGTATC 4861 TTTATAGTCC TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG TGATGCTCGT 4921 CAGGGGGGCG GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG TTCCTGGCCT 4981 TTTGCTGGCC TTTTGCTCAC ATGTT //



7. Troubleshooting

Problem Suggestion Few or no colonies obtained from LR reaction after transformation while the transformation control gave colonies

Incorrect antibiotic used for the selection of transformants - Check antibiotic marker of the destination vector used and

for transformants on new LB/antibiotic plates If you use the “fast protocol”, the LR reactions are not treated with Proteinase K. This causes a reduction of transformants. - Switch to the High Confidence protocol Clonase™ enzyme mix failure (inactive enzymes or didn’t use suggested amount) - Test another aliquot of the LR Clonase - Ensure storage of Clonase at –80°C - Do not freeze/thaw Clonase more than 10x - Use recommended amounts The destination vector used did not have the correct att sites - Always use destination vectors with attL1 and attL2

recombination sites Incorrect Clonase™ enzyme mix - Always use LR Clonase™ from Invitrogen to perform the

shuttling reaction Biased vector concentrations, especially too much Gateway Full ORF Clone DNA - Ensure equal molar amounts of Gateway Full ORF Clone

and destination vector Different colony sizes after transformation and plating

Loss of plasmid during bacterial growth - Re-incubate on selective plates at 30°C - Control for insert re-arrangements during the experiment - Use E. coli strains that stabilize unusual DNA structures

and repeat cloning (e.g. SURE from Stratagene) Small colonies can originate from Gateway Full ORF Clone plasmid DNA that co-transforms with the destination construct - Reduce the amount of Gateway Full ORF Clone to as low

as 25 ng/20 µl LR reaction Small colonies can originate from depletion of antibiotic on the plate (‘satellite colonies’ = small colonies surrounding a large colony) - Phenomenon seen primarily when using ampicillin

selection, as this antibiotic is consumed by bacteria. Replace ampicillin by carbenicillin (an analogon that is not used up by E. coli



High background in the Gateway Full ORF Clone reaction

LR reaction transformed into an F′ episome E. coli containing the ccdA gene - Use E. coli without F′ episome Inactive ccdB gene on the destination vector - Always propagate destination vectors in DB3.1 E. coli and

add 25ng/µl chloramphenicol to medium and plates used - Verify the destination vector before use (by restriction digest

or sequencing) One of the solutions is contaminated with a second plasmid - Verify your solutions by doing mock transformations with

the individual solutions - Make up new solutions Competent bacteria already carry a plasmid - Plate mock transformation directly

No colonies/less than 10 colonies after transformation

Problems with - Transformation - Competent bacteria - Insufficient amount of transformation plated



8. Literature

8.1. General

Bernard P., and M. Couturier. Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. J Mol Biol 1992, 226(3), 735-745.

Bernard P., K.E. Kezdy, L.V. Melderen, J. Steyaert, L. Wyns, M.L. Pato, P.N. Higgins, and M. Couturier. The F plasmid CcdB protein induces efficient ATP-dependent DNA cleavage by gyrase. J Mol Biol 1993, 234, 534-541.

Bushman W., J.F. Thompson, L. Vargas, and A. Landy. Control of directionality in lambda site-specific recombination. Science 1985, 230, 906-911.

Hartley J.L., G.F. Temple, and M.A. Brasch. DNA cloning using in vitro site-specific recombination. Genome Research 2000, 10, 1788-1795.

Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 1987, 15, 8125-8148.

Kozak M. Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes. Proc Natl Acad Sci USA 1990, 87, 8301-8305.

Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biology 1991, 115, 887-903.

Lander E., et al. Initial sequencing and analysis of the human genome. Nature 2001, 409, 860-921.

Landy A. Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Ann Rev Biochem 1989, 58, 913-949.

Miki T., J.A. Park, K. Nagao, N. Murayama, and T. Horiuchi. Control of segregation of chromosomal DNA by sex factor F in Escherichia coli. Mutants of DNA gyrase subunit a suppress letD (ccdB) product growth inhibition. J Mol Biol 1992, 225, 39-52.

Nomura N., T. Nagase, N. Miyajima, T. Sazuka, A. Tanaka, S. Sato, N. Seki, Y. Kawarabayasi, K. Ishikawa, and S. Tabata. Prediction of the coding sequences of unidentified human genes. II. The coding sequences of 40 new genes (KIAA0041-KIAA0080) deduced by analysis of cDNA clones from human cell line KG-1. DNA Res 1994, 1, 251-262.

Orosz A., I. Boros, and P. Venetianer. Analysis of the complex transcription termination region of the Escherichia coli rrnB Gene. Eur J Biochem 1991, 201, 653-659.

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor (N.Y.)

Shine J., and L. Dalgarno. Terminal-sequence analysis of bacterial ribosomal RNA. Correlation between the 3'-terminal-polypyrimidine sequence of 16-S RNA and translational specificity of the ribosome. Eur J Biochem 1975, 57(1), 221-230.

Straussberg R., et al. Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. PNAS 2002, 99, 16899-16903.

Venter J.C., et al. The sequence of the human genome. Science 2001, 291, 1304-1351. Weisberg R.A., and A. Landy. Site-specific recombination in phage lambda. In Lambda II, R.A.

Weisberg, ed. (Cold Spring Harbor, NY: Cold Spring Harbor Press) 1983, pp. 211-250. Wiemann S., B. Weil, R. Wellenreuther, J. Gassenhuber, S. Glassl, W. Ansorge, M. Bocher, H.

Blocker, S. Bauersachs, H. Blum, J. Lauber, A. Dusterhoft, A. Beyer, K. Kohrer, N. Strack, H.W. Mewes, B. Ottenwalder, B. Obermaier, J. Tampe, D. Heubner, R. Wambutt, B. Korn, M. Klein, and A. Poustka. Toward a catalog of human genes and proteins: sequencing and analysis of 500 novel complete protein coding human cDNAs. Genome Res 2001, 11, 422-435.

8.2. Applications

8.2.1. Expression in Bacteria

Braun P., Y. Hu, B. Shen, A. Halleck, M. Koundinya, E. Harlow, and J. LaBaer. Proteome-scale purification of human proteins from bacteria. Proc Natl Acad Sci USA 2002, 99(5), 2654-2659.

Hammarstrom M., N. Hellgren, S. van Den Berg, H. Berglund, and T. Hard. Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Science 2002, 11, 313-321.

Kulkarni G.V., and D.D. Deobagkar. A cytosolic form of aminopeptidase P from Drosophila melanogaster: molecular cloning and characterization. J Biochem 2002, 131(3), 445-452.



Kuma A., N. Mizushima, N. Ishihara, and Y. Ohsumi. Formation of the approximately 350-kDa Apg12-Apg5.Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for autophagy in yeast. J Biol Chem 2002, 277(21), 18619-18625.

Reina J., E. Lacroix, S.D. Hobson, G. Fernandez-Ballester, V. Rybin, M.S. Schwab, L. Serrano, C. Gonzalez. Computer-aided design of a PDZ domain to recognize new target sequences. Nature Structural Biology 2002, 9, 621-627.

Wang Y., J.A. Bruenn, S.F. Queener, and V. Cody. Isolation of rat dihydrofolate reductase gene and characterization of recombinant enzyme. Antimicrob Agents Chemother 2001, 45(9), 2517-2523.

Way G., N. Morrice, C. Smythe, and A.J. O'Sullivan. Purification and identification of secernin, a novel cytosolic protein that regulates exocytosis in mast cells. Mol Biol Cell 2002, 13(9), 3344-3354.

8.2.2. Yeast Expression

Brizuela L., P. Braun, and J. LaBaer. FLEXGene repository: from sequenced genomes to gene repositories for high-throughput functional biology and proteomics. Mol Biochem Parasitol 2001, 118(2), 155-165.

Courbard J., F. Frederic, J. Adelaide, J. Borg, D. Birnbaum, and V. Ollendorf. Interaction between two E3 ubiquitin ligases of different classes, CBLC and AIP4/ITCH. J Biol Chem 2002, 277, 45267-45275.

Davy A., P. Bello, N. Thierry-Mieg, P. Vaglio, J. Hitti, L. Doucette-Stamm, D. Thierry-Mieg, J. Reboul, S. Boulton, A.J. Walhout, O. Coux, and M. Vidal. A protein-protein interaction map of the Caenorhabditis elegans 26S proteasome. EMBO Rep 2001, 2(9), 821-828.

Finley R.L. Jr., H. Zhang, J. Zhong, and C.A. Stanyon. Regulated expression of proteins in yeast using the MAL61-62 promoter and a mating scheme to increase dynamic range. Gene 2002, 285(1-2), 49-57.

Funk M., R. Niedenthal, D. Mumberg, K. Brinkmann, V. Ronicke, and T. Henkel. Vector systems for the heterologous expression of proteins in Saccharomyces cerevisiae. Methods Enzymol 2002, 350, 248-257.

Gavin A.C., M. Bosche, R. Krause, P. Grandi, M. Marzioch, A. Bauer, J. Schultz, J.M. Rick, A.M. Michon, C.M. Cruciat, M. Remor, C. Hofert, M. Schelder, M. Brajenovic, H. Ruffner, A. Merino, K. Klein, M. Hudak, D. Dickson, T. Rudi, V. Gnau, A. Bauch, S. Bastuck, B. Huhse, C. Leutwein, M.A. Heurtier, R.R. Copley, A. Edelmann, E. Querfurth, V. Rybin, G. Drewes, M. Raida, T. Bouwmeester, P. Bork, B. Seraphin, B. Kuster, G. Neubauer, and G. Superti-Furga. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 2002, 415, 141-147.

Stelzl, U., U. Worm, M. Lalowski, Chr. Haenig, F. H. Brembeck, H. Goehler, M. Stroedicke, M. Zenkner, A. Schoenherr, S. Koeppen, J. Timm, S. Mintzlaff, C. Abraham, N. Bock, S. Kietzmann, A. Goedde, E. Toksoz, A. Droege, S. Krobitsch, B. Korn, W. Birchmeier, H. Lehrach, and E. E. Wanker . A Human Protein-Protein Interaction Network: A Resource for Annotating the Proteome. Cell 2005, 122, Sept. 1st.

Walhout A.J., R. Sordella, X. Lu, J.L. Hartley, G.F. Temple, M.A. Brasch, N. Thierry-Mieg, and M. Vidal. Protein interaction mapping in C. elegans using proteins involved in vulval development. Science 2000, 287(5450), 116-22.

Walhout A.J., and M. Vidal. High-throughput yeast two-hybrid assays for large-scale protein interaction mapping. Methods 2001, 24(3), 297-306.

8.2.3. Baculo Virus Expression

Akgoz M., I. Azpiazu, V. Kalyanaraman, and N. Gautam. Role of the G protein gamma subunit in beta gamma complex modulation of phospholipase Cbeta Function. J Biol Chem 2002, 277(22), 19573-19578.

Giebler H.A., I. Lemasson, and J. Nyborg. p53 Recruitment of CREB binding protein mediated through phosphorylated CREB: a novel pathway of tumor suppressor regulation. Mol Cell Biol 2000, 20(13), 4849-4858.

Grand-Perret T., A. Bouillot, A. Perrot, S. Commans, M. Walker, and M. Issandou. SCAP ligands are potent new lipid-lowering drugs. Nature Medicine 2001, 7, 1332-1338.

Iwai T., et al. Molecular cloning and characterization of a Novel UDP-GlcNAc:GalNAc-peptide b3Gn-T6), an enzyme synthesizing the core 3 structure of O-glycans. J Biol Chem 2002, 277(15), 12802-12809.



Katagiri Y., and K.C. Ingham. Enhanced production of green fluorescent fusion proteins in a baculovirus expression system by addition of secretion signal. BioTechniques 2002, 33(1), 24-26.

Wu X., S.R. Webster, and J. Chen. Characterization of tumor-associated Chk2 mutations. J Biol Chem 2001, 276(4), 2971-2974.

8.2.4. Expression in Plant Cells

Karimi M., D. Inze, and A. Depicker. Gateway® vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 2002, 7(5), 193-195.

Kersten B., L. Burkle, E.J. Kuhn, P. Giavalisco, Z. Konthur, A. Lueking, G. Walter, H. Eickhoff, and U. Schneider. Largescale plant proteomics. Plant Mol Biol 2002, 48, 133-141.

Koiwa H., A.W. Barb, L. Xiong, F. Li, M.G. McCully, B.H. Lee, I. Sokolchik, J. Zhu, Z. Gong, M. Reddy, A. Sharkhuu, Y. Manabe, S. Yokoi, J.K. Zhu, R.A. Bressan, and P.M. Hasegawa. C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development. Proc Natl Acad Sci USA 2002, 99(16), 10893-10898.

Wolucka B.A., G. Persiau, J. Van Doorsselaere, M.W. Davey, H. Demol, J. Vandekerckhove, M. Van Montagu, M. Zabeau, and W. Boerjan. Partial purification and identification of GDP-mannose 3",5"-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway. Proc Natl Acad Sci USA 2001, 98(26), 14843-14848.

8.2.5. Mammalian Cell Line Expression

Blin-Wakkach C., F. Lezot, S. Ghoul-Mazgar, D. Hotton, S. Monteiro, C. Teillaud, L. Pibouin, S. Orestes-Cardoso, P. Papagerakis, M. Macdougall, B. Robert, and A. Berdal. Endogenous Msx1 antisense transcript: in vivo and in vitro evidences, structure, and potential involvement in skeleton development in mammals. Proc Natl Acad Sci USA 2001, 98(13), 7336-7341.

Gomes M.D., S.H. Lecker, R.T. Jagoe, A. Navon, and A.L. Goldberg. Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy. Proc Natl Acad Sci USA 2001, 98(25), 14440-14445.

Kudo T., T. Iwai, T. Kubota, H. Iwasaki, Y. Takayma, T. Hiruma, N. Inaba, Y. Zhang, M. Gotoh, A. Togayachi, and H. Narimatsu. Molecular cloning and characterization of a covel UDP-Gal:GalNAc(alpha) peptide beta 1,3-galactosyltransferase (C1Gal-T2), an enzyme synthesizing a core 1 structure of O-glycan. J Biol Chem 2002, 277(49), 47724-47731.

Lao G., D. Polayes, J.L. Xia, F.R. Bloom, F. Levine, and J. Mansbridge. Overexpression of trehalose synthase and accumulation of intracellular trehalose in 293H and 293FTetR:Hyg cells. Cryobiology 2001, 43(2), 106-113.

Liu J., F. Yao, R. Wu, M. Morgan, A. Thorburn, R. Finley, and Y.Q. Chen. Mediation of the DCC apoptotic signal by DIP13alpha. J Biol Chem 2002, 277(29), 26281-26285.

Loftus S., D. Larson, L. Baxter, A. Antonellis, Y. Chen, X. Wu, Y. Jiang, M. Bittner, J. Hammer, and W. Pavan. Mutation of melanosome protein RAB38 in chocolate mice. Proc Natl Acad Sci USA 2002, 99(7), 4471-4476.

Pandey A., N. Ibarrola, I. Kratchmarova, M.M. Fernandez, S.N. Constantinescu, O. Ohara, S. Sawasdikosol, H.F. Lodish, and M. Mann. A novel Src homology 2 domain-containing molecule, Src-like adapter protein-2 (SLAP-2), which negatively regulates T cell receptor signaling. J Biol Chem 2002, 277(21), 19131-19138.

8.2.6. Miscellaneous

Blaszczyk J., J.E. Tropea, M. Bubunenko, K.M. Routzahn, D.S. Waugh, D.L. Court, and X. Ji. Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage. Structure 2001, 9(12), 1225-1236.

Bloom F.R., P. Price, G. Lao, J.L. Xia, J.H. Crowe, J.R. Battista, R.F. Helm, S. Slaughter, and M. Potts. Engineering mammalian cells for solidstate sensor applications. Biosens Bioelectron 2001, 16(7-8), 603-608.

Brizuela L., P. Braun, and J. LaBaer. FLEXGene repository: from sequenced genomes to gene repositories for highthroughput functional biology and proteomics. Mol Biochem Parasitol 2001, 118(2), 155-165.



Hammarstrom M., N. Hellgren, S. van Den Berg, H. Berglund, and T. Hard. Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Science 2002, 11(2), 313-321.

Hoover D.M., and J. Lubkowski. DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res 2002, 30(10), e43.

Jakobs A, Koehnke J, Himstedt F, Funk M, Korn B, Gaestel M, Niedenthal R. Ubc9 fusion-directed SUMOylation (UFDS): a method to analyze function of protein SUMOylation. Nat Methods. 2007, 4, 245-50.

Jhoti H. High-throughput structural proteomics using x-rays. Trends Biotechnology 2001, 19(10), 67-71.

Ko M.S. Embryogenomics: developmental biology meets genomics. Trends Biotechnology 2001, 19(12), 511-518.

Matthews L.R., P. Vaglio, J. Reboul, H. Ge, B.P. Davis, J. Garrels, S. Vincent, and M. Vidal. Identification of potential interaction networks using sequence-based searches for conserved protein-protein interactions or "interologs". Genome Res 2001, 11(12), 2120-2126.

Monchois V., R. Vincentelli, C. Deregnaucourt, J. Claverie, and C. Abergel. Proficient target selection in structural genomics by in-vitro protein expression. Biochemica (Roche) 2002, 1, 22.

Ohara O., and G. Temple. Directional cDNA library construction assisted by the in vitro recombination reaction. Nucleic Acids Res 2001, 29(4), e22.

Ohara O., T. Nagase, G. Mitsui, H. Kohga, R. Kikuno, S. Hiraoka, Y. Takahashi, S. Kitajima, Y. Saga, and H. Koseki. Characterization of size-fractionated cDNA libraries generated by the in vitro recombination-assisted method. DNA Res 2002, 9(2), 47-57.

Reboul J., P. Vaglio, N. Tzellas, N. Thierry-Mieg, T. Moore, C. Jackson, T. Shin-i, Y. Kohara, D. Thierry-Mieg, J. Thierry-Mieg, H. Lee, J. Hitti, L. Doucette-Stamm, J.L. Hartley, G.F. Temple, M.A. Brasch, J. Vandenhaute, P.E. Lamesch, D.E. Hill, and M. Vidal. Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans. Nat Genet 2001, 27(3), 332-336.

Simpson J.C., V.E. Neubrand, S. Wiemann, and R. Pepperkok. Illuminating the human genome. Histochem Cell Biol 2001, 115(1), 23-29.

Simpson J.C., R. Wellenreuther, A. Poustka, R. Pepperkok, and S. Wiemann. Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing. EMBO Rep 2000, 1(3), 287-292.

Walter G., K. Bussow, A. Lueking, and J. Glokler. High-throughput protein arrays: prospects for molecular diagnostics. Trends Mol Med 2002, 8(6), 250-253.

Wiemann S., B. Weil, R. Wellenreuther, J. Gassenhuber, S. Glassl, W. Ansorge, M. Bocher, H. Blocker, S. Bauersachs, H. Blum, J. Lauber, A. Dusterhoft, A. Beyer, K. Kohrer, N. Strack, H.W. Mewes, B. Ottenwalder, B. Obermaier, J. Tampe, D. Heubner, R. Wambutt, B. Korn, M. Klein, and A. Poustka. Toward a catalog of human genes and proteins: sequencing and analysis of 500 novel complete protein coding human cDNAs. Genome Res 2001, 11(3), 422-435.

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