LOCUS AW561511 568 bp mRNA linear EST 08-MAR-2000 DEFINITION ga81e06.y1 Moss EST library PPU Physcomitrella patens cDNA clone PEP_SOURCE_ID:PPU150311 5' similar to gb:emb|Z24678.1|VFNUREPRA V.faba guanine nucleotide regulatory (PLANT), mRNA sequence. ACCESSION AW561511 VERSION AW561511.1 DBLINK BioSample: SAMN00157056 KEYWORDS EST. SOURCE Physcomitrium patens ORGANISM Physcomitrium patens Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Bryophyta; Bryophytina; Bryopsida; Funariidae; Funariales; Funariaceae; Physcomitrium. REFERENCE 1 (bases 1 to 568) AUTHORS Quatrano,R., Bashiardes,S., Cove,D., Cuming,A., Knight,C., Clifton,S., Marra,M., Hillier,L., Pape,D., Martin,J., Wylie,T., Underwood,K., Theising,B., Allen,M., Bowers,Y., Person,B., Swaller,T., Steptoe,M., Gibbons,M., Harvey,N., Ritter,E., Jackson,Y., McCann,R., Waterston,R. and Wilson,R. TITLE Leeds/Wash U Moss EST Project JOURNAL Unpublished COMMENT Contact: Ralph Quatrano Leeds/Wash U Moss EST Project Washington University School of Medicine 4444 Forest Park Parkway, Box 8501, St. Louis, MO 63108, USA Tel: 314 286 1800 Fax: 314 286 1810 Email: est@watson.wustl.edu Libraries were constructed by Dr. Stavros Bashiardes as part of the Physcomitrella EST program (PEP) at the Univ. of Leeds (UK) and Washington Univ. in St. Louis (USA) DNA sequencing by: Washington University Genome Sequencing Center For information on obtaining a clone please contact: Celia Knight (c.d.knight@leeds.ac.uk) Seq primer: -40RP from Gibco High quality sequence stop: 412. FEATURES Location/Qualifiers source 1..568 /organism="Physcomitrium patens" /mol_type="mRNA" /db_xref="taxon:3218" /clone="PEP_SOURCE_ID:PPU150311" /tissue_type="protonemata: 7 day old tissue ammonium-grown" /clone_lib="SAMN00157056 Moss EST library PPU" /lab_host="DH10B" /note="Vector: pBluescript SK-; Site_1: EcoRI; Site_2: XhoI; Construction of the cDNA library was carried out using Stratagenes 'UniZAP - cDNA synthesis kit'. cDNA was constructed using an oligo dT primer/linker that contains a XhoI site within it. Following ds cDNA synthesis, EcoRI adapters were ligated to the blunt ends and sample was digested with XhoI. The result is cDNA with an EcoRI sticky end on one side and a XhoI sticky end on the other. This cDNA was ligated directionally in UniZAP arms. The vector is designed containing the pBluescript sequence as well as lambda DNA and cDNA is cloned within this pBluescript sequence. The vector was then packaged using Gold gigapackaging extracts. Library was grown in XLIBlue MRF' cells and amplified. The library was excised by mass excision using Stratagens 'Mass excision kit' that uses exassist as a helper phage that releases the pBluescript sequence and circularises it as single stranded plasmids that are then packaged (by helper phage) and secreted out of the host cell as phagemids. SOLR cells were transformed with phagemids and the library was plated out on LB-amp plates to select for transformants. Approximately 1,000,000 colonies were grown and recovered. The double stranded plasmid library was recovered by using Quiagen Midi prep kit. 2 micro grams of each library were used to transform DH10B cells by electroporation." BASE COUNT 134 a 152 c 150 g 131 t ORIGIN 1 aggctctgct gcttcctccc cttcccccct tccctgcaga ttggcgtcgc ctcgcgggct 61 cgtgttgagc ctttgtagat cgccgcacag ctccgagtca tggctttgcc agggcagcaa 121 acccccgaca actgcccaag ctttaagctc gtcattgtcg gagacggagg cacaggaaag 181 accacctttg tgaagcgtca tttgactggt gaattcgaaa aaagatacga acctaccatc 241 ggtgtggagg ttcacccact tgacttcttc accaactgcg ggcgtatccg attttactgc 301 tgggataccg ctggacaaga gaaatttgga gggcttcgtg atggctacta cattcacggt 361 caatgtgcca tcatcatgtt cgatgttact gctcggctta cgtataagaa cgtcccgaca 421 tggcatcgtg acctttgcan ggtgtgtgag aacatcccca tcgtgctgtg cgggaacaag 481 gtggacgtga agaacaggca ggtgaaagcg aagcaagtga ctgtgcacag gaaaacagaa 541 cctgccatac taccagattt ctgcgaag //