Microsoft word - pgk-gb2-cm-frt-v2.0-page1to3.doc

Gene Bridges GmbH Im Neuenheimer Feld 584 69120 Heidelberg, Germany Tel + 49 (0)6221 13708 11 Fax + 49 (0)6221 13708 29 Email: contact@genebridges.com www.genebridges.com CONTENTS
1 Eppendorf tubes + manual
1. FRT-cm-FRT: PCR template (50 ng/µl, 20µl) 2. This manual Store tube at -20°C

Please read
The products listed in this manual are for research purposes only. They are not designed for
diagnostic or therapeutic use in humans, animals or plants. The Red®/ET® recombination
technology is the intellectual property of Gene Bridges GmbH.

Conditions of use
3.1 Purchaser will not manufacture, copy, reproduce, transmit, distribute, sell, lease, transfer, or improve upon the MATERIALS without prior written consent from GENE BRIDGES. 3.2 All MATERIALS related to the technology must be purchased from GENE BRIDGES or its authorized distributors. Use of any of the stated products from a source other than GENE BRIDGES will exempt GENE BRIDGES from any and all liabilities and warranties. 3.3 All MATERIALS purchased by research organizations, universities and other non-profit organizations may not be used for any commercial purpose. These MATERIALS are to be used for research purposes only. The MATERIALS may not be used to provide a commercial or non-commercial service, of any kind. 3.4 The purchase of MATERIALS by a private consumer is neither intended nor permitted. Short Description:

“FRT-cm-FRT” cassette is designed to allow chloramphenicol selection in The prokaryotic promoter gb2 driving the gene for chloramphenicol resistance is a slightly modified version of the Em7 promoter; it mediates higher transcription efficiency than the generally used Tn5 promoter. A synthetic polyadenylation signal terminates the chloramphenicol expression. The cassette is flanked by FRT sites for later excision by Flp-recombinase. Unique NotI and XhoI sites flank the cassette for convenient cloning with restriction Using the provided PCR template one can easily create a FRT-cm-FRT cassette flanked by any other restriction sites to clone the cassette into the vector of choice. The restriction sites can be introduced by adding the corresponding sequence in the PCR primer. The template can easily be used to engineer the E. coli genome by Red®/ET® The “FRT-cm-FRT cassette” is not linear but plasmid based (3302 bp in size). Due to its R6K origin the plasmid cannot replicate in most E. coli strains. The PCR product can therefore be used directly for downstream applications At least 20 PCR reactions can be performed using 1 µl per reaction as chloramphenicol
AATTAACCCTCACTAAAGG GCGGCCGC GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC ATTCTACCGG GTAGGGGAGG CGCTTTTCCC AAGGCAGTCT GGAGCATGCG CTTTAGCAGC CCCGCTGGGC ACTTGGCGCT 142 ACACAAGTGG CCTCTGGCCT CGCACACATT CCACATCCAC CGGTAGGCGC CAACCGGCTC CGTTCTTTGG 212 TGGCCCCTTC GCGCCACCTT CCACTCCTCC CCTAGTCAGG AAGTTCCCCC CCGCCCCGCA GCTCGCGTCG 282 TGCAGGACGT GACAAATGGA AGTAGCACGT CTCACTAGTC TCGTGCAGAT GGACAGCACC GCTGAGCAAT 352 GGAAGCGGGT AGGCCTTTGG GGCAGCGGCC AATAGCAGCT TTGCTCCTTC GCTTTCTGGG CTCAGAGGCT 422 GGGAAGGGGT GGGTCCGGGG GCGGGCTCAG GGGCGGGCTC AGGGGCGGGG CGGGCGCCCG AAGGTCCTCC 492 GGAGGCCCGG CATTCTGCAC GCTTCAAAAG CGCACGTCTG CCGCGCTGTT CTCCTCTTCC TCATCTCCGG 562 GCCTTTCGAC C TGCAGC AGCACGTGTT GACAATTAAT CATCGGCATA GTATATCGGC ATAGTATAAT 629 ACGACAAGGT GAGGAACTAA ACC ATG GAG AAA AAA ATC ACT GGA TAT ACC ACC GTT GAT ATA TCC 1 Me t Gl u Ly s Ly s I l e Thr Gl y Ty r Th r Thr Va l Asp I l e Ser 694 CAA TGG CAT CGT AAA GAA CAT TTT GAG GCA TTT CAG TCA GTT GCT CAA TGT ACC TAT AAC CAG 15 Gl n Tr p Hi s A r g Ly s Gl u Hi s Phe Gl u A l a Phe Gl n Se r Va l A l a Gl n Cy s Th r Ty r Asn Gl n 757 ACC GTT CAG CTG GAT ATT ACG GCC TTT TTA AAG ACC GTA AAG AAA AAT AAG CAC AAG TTT TAT Thr Val Gl n Leu Asp I l e Thr Al a Phe Leu Ly s Thr Val Ly s Ly s Asn Ly s Hi s Ly s Phe Ty r 820 CCG GCC TTT ATT CAC ATT CTT GCC CGC CTG ATG AAT GCT CAT CCG GAA TTC CGT ATG GCA ATG 57 P r o A l a Phe I l e Hi s I l e Leu A l a A r g Leu Me t Asn A l a Hi s P r o Gl u Phe A r g Me t A l a Me t 883 AAA GAC GGT GAG CTG GTG ATA TGG GAT AGT GTT CAC CCT TGT TAC ACC GTT TTC CAT GAG CAA Ly s Asp Gly Gl u Leu Val I l e Tr p Asp Ser Val Hi s Pr o Cy s Ty r Thr Val Phe Hi s Gl u Gl n 946 ACT GAA ACG TTT TCA TCG CTC TGG AGT GAA TAC CAC GAC GAT TTC CGG CAG TTT CTA CAC ATA Thr Gl u Thr Phe Ser Ser Leu Tr p Ser Gl u Ty r Hi s Asp Asp Phe Ar g Gl n Phe Leu Hi s I l e 1009 TAT TCG CAA GAT GTG GCG TGT TAC GGT GAA AAC CTG GCC TAT TTC CCT AAA GGG TTT ATT GAG 120 Ty r Se r Gl n Asp Va l A l a Cy s Ty r Gl y Gl u Asn Leu A l a Ty r Phe P r o Ly s Gl y Phe I l e Gl u 1072 AAT ATG TTT TTC GTC TCA GCC AAT CCC TGG GTG AGT TTC ACC AGT TTT GAT TTA AAC GTG GCC 141 Asn Me t Phe Phe Va l Se r A l a Asn P r o Tr p Va l Se r Phe Th r Se r Phe Asp Leu Asn Va l A l a 1135 AAT ATG GAC AAC TTC TTC GCC CCC GTT TTC ACC ATG GGC AAA TAT TAT ACG CAA GGC GAC AAG 162 Asn Me t Asp Asn Phe Phe A l a P r o Va l Phe Th r Me t Gl y Ly s Ty r Ty r Th r Gl n Gl y Asp Ly s 1198 GTG CTG ATG CCG CTG GCG ATT CAG GTT CAT CAT GCC GTT TGT GAT GGC TTC CAT GTC GGC AGA 183 Va l Leu Me t P r o Leu A l a I l e Gl n Va l Hi s Hi s A l a Va l Cy s Asp Gl y Phe Hi s Va l Gl y A r g 1261 ATG CTT AAT GAA TTA CAA CAG TAC TGC GAT GAG TGG CAG GGC GGG GCG TAAGCGGGACTCTGGGGTT 204 Me t Leu Asn Gl u Leu Gl n Gl n Ty r Cy s Asp Gl u Tr p Gl n Gl y Gl y A l a • • • 1328 CGAATAAAGACCGACCAAGCGAC GTC TGA GAGCTCCCTG GCGAATTCGG TACCAATAAA AGAGCTTTAT 1397 TTTCATGATC TGTGTGTTGG TTTTTGTGTG CGGCGCG GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC C TCGAG Please take into consideration that the sequence given above does not reflect thecomplete plasmid but refers to the functional cassette.

Source: http://www.genebridges.com/gb/pdf/A006-FRT-cm-FRT.pdf