LOCUS       Exported               10649 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector EN.Apple, complete sequence.
ACCESSION   HM771700
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10649)
  AUTHORS   Chen SX, Osipovich AB, Ustione A, Potter LA, Hipkens S, Gangula R, 
            Yuan W, Piston DW, Magnuson MA.
  TITLE     Quantification of factors influencing fluorescent protein expression
            using RMCE to generate an allelic series in the ROSA26 locus in mice
  JOURNAL   Dis Model Mech 4 (4), 537-547 (2011)
  PUBMED    21324933
REFERENCE   2  (bases 1 to 10649)
  AUTHORS   Chen SX, Osipovich AB, Magnuson MA.
  TITLE     Direct Submission
  JOURNAL   Submitted (16-JUL-2010) Molecular Physiology and Biophysics, 
            Vanderbilt University, 9475 MRB IV, 2214 Garland Avenue, Nashville, 
            TN 37232, USA
REFERENCE   3  (bases 1 to 10649)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10649
                     /organism="Cloning vector EN.Apple"
                     /mol_type="other DNA"
                     /db_xref="taxon:1052599"
     promoter        complement(13..31)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(52..68)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    76..92
                     /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(100..130)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    145..166
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(454..1042)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1213..2073)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(2074..2178)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2253..2708)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     2850..2866
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2873..2891
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    2906..2939
                     /label=Lox66 site
                     /note="Lox66 site"
     protein_bind    complement(2906..2939)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     regulatory      2957..7037
                     /regulatory_class="promoter"
                     /note="Rosa 26 promoter"
     5UTR           7043..7101
                     /label=translational enhancer
                     /note="translational enhancer"
     5UTR           7050..7092
                     /label=Xenopus globin 5-UTR
                     /note="translational enhancer from the 5-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             7103..7813
                     /codon_start=1
                     /product="mApple"
                     /label=mApple
                     /note="red fluorescent protein"
                     /protein_id="AEM37572.1"
                     /translation="MVSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEA
                     FQTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNF
                     EDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALK
                     SEIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     regulatory      7827..8712
                     /regulatory_class="polyA_signal_sequence"
                     /note="rabbit beta globin intron poly A signal"
     intron          7841..8413
                     /label=-beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     polyA_site      8610..8665
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     misc_feature    8728..8761
                     /label=FRT site
                     /note="FRT site"
     protein_bind    8728..8761
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     regulatory      8809..9401
                     /regulatory_class="promoter"
                     /note="PGK promoter"
     promoter        8825..9324
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     promoter        9336..9383
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9402..10205
                     /codon_start=1
                     /product="Neo"
                     /label=Neo
                     /protein_id="AEM37573.1"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     regulatory      10219..10520
                     /regulatory_class="polyA_signal_sequence"
                     /note="rabbit beta globin poly A signal"
     polyA_signal    10243..10467
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    10525..10558
                     /label=FRT site
                     /note="FRT site"
     protein_bind    10525..10558
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    complement(10611..10644)
                     /label=Lox2272 site
                     /note="Lox2272 site"
     protein_bind    complement(10611..10644)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites."