LOCUS       Exported                4236 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pEN-2xChimera, complete sequence.
ACCESSION   KY489664
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4236)
  AUTHORS   Durr J, Papareddy R, Nakajima K, Gutierrez-Marcos J.
  TITLE     Highly efficient heritable targeted deletions of gene clusters and 
            non-coding regulatory regions in Arabidopsis using CRISPR/Cas9
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4236)
  AUTHORS   Durr J, Gutierrez-Marcos J.
  TITLE     Direct Submission
  JOURNAL   Submitted (19-JAN-2017) School of Life Sciences, University of 
            Warwick, Gibbet Hill Campus, Coventry, West Midlands CV4 7AL, United
            Kingdom
REFERENCE   3  (bases 1 to 4236)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4236
                     /organism="Cloning vector pEN-2xChimera"
                     /mol_type="other DNA"
                     /db_xref="taxon:2107236"
     regulatory      33..42
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     regulatory      37..46
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     misc_recomb     52..151
                     /label=attL1
                     /note="attL1"
     protein_bind    52..151
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      152..539
                     /regulatory_class="promoter"
                     /note="U6-26"
     misc_feature    558..674
                     /label=sgRNA scaffold
                     /note="sgRNA scaffold"
     misc_RNA        558..633
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(645..669)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      681..1068
                     /regulatory_class="promoter"
                     /note="U6-26"
     misc_feature    1085..1201
                     /label=sgRNA scaffold
                     /note="sgRNA scaffold"
     misc_RNA        1085..1160
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1172..1196)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_recomb     1226..1325
                     /label=attL2
                     /note="attL2"
     protein_bind    complement(1226..1325)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1361..1379)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1397..1413)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1421..1437
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(1445..1475)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1490..1511
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(1799..2387)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2558..3418)
                     /codon_start=1
                     /gene="ampR"
                     /product="AmpR"
                     /label=ampR
                     /protein_id="AVI59150.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(2558..3418)
                     /gene="ampR"
                     /label=ampR
     promoter        complement(3419..3523)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3601..4056)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     4197..4213
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4220..2
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"