LOCUS       2t_insert                226 bp ds-DNA     linear       09-AUG-2020
DEFINITION  Cloning vector pGEM-7Zf(+).
ACCESSION   X65310
VERSION     X65310.2  GI:8216950
KEYWORDS    beta-lactamase; bla gene; cloning vector; lacZ gene; multiple
            cloning site; phage f1 region; promoter.
SOURCE      Cloning vector pGEM-7Zf(+)
  ORGANISM  Cloning vector pGEM-7Zf(+) other sequences; artificial sequences;
            vectors.
REFERENCE   1
  AUTHORS   Technical,Services.
  TITLE     Direct Submission
  JOURNAL   Submitted (23-MAR-1992) Technical Services, Promega Corporation,
            2800 Woods Hollow Road, Madison, Wi 53711-5399, USA
  REMARK    revised by [2]
REFERENCE   2
  AUTHORS   Technical,Services.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-MAY-1993) Technical Services, Promega Corporation,
            2800 Woods Hollow Road, Madison, Wi 53711-5399, USA
  REMARK    revised by [3]
REFERENCE   3  (bases 1 to 2997)
  AUTHORS   Technical,Services.
  TITLE     Direct Submission
  JOURNAL   Submitted (22-MAY-2000) Technical Services, Promega Corporation,
            2800 Woods Hollow Road, Madison, Wi 53711-5399, USA
COMMENT     Synthetic fragment: HindIII site; BsaI site (ggtctcn1/5); sgRNA
            (for Cas9) targeting Arabidopsis KN (At1g08560; protospacer:
            GTTTCTCACGCGAGCAATTC); Arabidopsis tRNA-alanine coding sequence;
            protospacer segment of sgRNA targeting Arabidopsis KEU (At1g12360;
            GATGTGACCAACCAGTTCCT); BsaI site (5/1ngagacc); EcoRI site. The
            fragment was inserted into the HindIII and EcoRI cloning sites of
            pGEM7Zf to produce pGEM-2t. The plasmid is intended to be used as
            template in PCR reactions designed to produce insert fragments for
            assembling multicistronic CRISPR RNAs in the binary vectors
            described by Xing and colleagues (2014, doi:
            10.1186/s12870-014-0327-y) or derived T-DNA vectors.
COMMENT     
COMMENT     ApEinfo:methylated:1
FEATURES             Location/Qualifiers
     misc_feature    7..12
                     /label=BsaI
                     /ApEinfo_fwdcolor="#cccccb"
                     /ApEinfo_revcolor="#cccccb"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    complement(215..220)
                     /label=BsaI(1)
                     /ApEinfo_label="BsaI"
                     /ApEinfo_fwdcolor="#cccccb"
                     /ApEinfo_revcolor="#cccccb"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    complement(221..226)
                     /label=EcoRI
                     /ApEinfo_fwdcolor="#cccccb"
                     /ApEinfo_revcolor="#cccccb"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    complement(1..6)
                     /label=HindIII
                     /ApEinfo_fwdcolor="#cccccb"
                     /ApEinfo_revcolor="#cccccb"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    17..36
                     /label=Protospacer KN (At1g08560)
                     /ApEinfo_fwdcolor="#cccccb"
                     /ApEinfo_revcolor="#cccccb"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    190..209
                     /label=Protospacer KEU (At1g12360)
                     /ApEinfo_fwdcolor="#cccccb"
                     /ApEinfo_revcolor="#cccccb"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    113..189
                     /label=tRNA-alanine coding sequence
                     /ApEinfo_fwdcolor="#cccccb"
                     /ApEinfo_revcolor="#cccccb"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    18..112
                     /label=sgRNA
                     /ApEinfo_fwdcolor="#cccccb"
                     /ApEinfo_revcolor="#cccccb"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    17..17
                     /label=sgRNA 5'end
                     /ApEinfo_fwdcolor="#ffff00"
                     /ApEinfo_revcolor="#ffff00"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    190..190
                     /label=sgRNA 5'end(1)
                     /ApEinfo_label="sgRNA 5'end"
                     /ApEinfo_fwdcolor="#ffff00"
                     /ApEinfo_revcolor="#ffff00"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
ORIGIN
        1 aagcttggtc tcgattGTTT CTCACGCGAG CAATTCgttt tagagctaga aatagcaagt
       61 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcaacaaagc
      121 accagtggtc tagtggtaga atagtaccct gccacggtac agacccgggt tcgattcccg
      181 gctggtgcaG ATGTGACCAA CCAGTTCCTg tttagagacc gaattc 
//
