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  JOURNAL   Unpublished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
//