LOCUS       Exported                7032 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Mammalian expression vector pNBioSec, complete sequence.
ACCESSION   EU082004
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7032)
  AUTHORS   Mize GJ, Harris JE, Takayama TK, Kulman JD.
  TITLE     Regulated expression of active biotinylated G-protein coupled 
            receptors in mammalian cells
  JOURNAL   Protein Expr. Purif. 57 (2), 280-289 (2008)
  PUBMED    18042400
REFERENCE   2  (bases 1 to 7032)
  AUTHORS   Mize GJ, Harris JE, Takayama TK, Kulman JD.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-AUG-2007) Department of Biochemistry, University of 
            Washington, N.E. Pacific Street, Seattle, WA 98195, USA
REFERENCE   3  (bases 1 to 7032)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7032
                     /organism="Mammalian expression vector pNBioSec"
                     /mol_type="other DNA"
                     /db_xref="taxon:478810"
     regulatory      7..439
                     /regulatory_class="promoter"
                     /note="tetracycline-responsive promoter"
     protein_bind    15..285
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     protein_bind    15..33
                     /label=TetR binding site
                     /bound_moiety="TetR"
                     /note="tetO repeat"
     protein_bind    57..75
                     /label=TetR binding site
                     /bound_moiety="TetR"
                     /note="tetO repeat"
     protein_bind    99..117
                     /label=TetR binding site
                     /bound_moiety="TetR"
                     /note="tetO repeat"
     protein_bind    141..159
                     /label=TetR binding site
                     /bound_moiety="TetR"
                     /note="tetO repeat"
     protein_bind    183..201
                     /label=TetR binding site
                     /bound_moiety="TetR"
                     /note="tetO repeat"
     protein_bind    225..243
                     /label=TetR binding site
                     /bound_moiety="TetR"
                     /note="tetO repeat"
     protein_bind    267..285
                     /label=TetR binding site
                     /bound_moiety="TetR"
                     /note="tetO repeat"
     promoter        318..356
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      320..439
                     /regulatory_class="promoter"
                     /note="minimal CMV promoter"
     regulatory      342..349
                     /regulatory_class="TATA_box"
     regulatory      477..485
                     /regulatory_class="ribosome_binding_site"
                     /note="Kozak site"
     misc_feature    570..608
                     /label=biotin acceptor peptide (BioTag)-encoding
                     /note="biotin acceptor peptide (BioTag)-encoding"
     misc_feature    609..628
                     /note="multiple cloning site; MCS"
     misc_feature    722..1302
                     /note="encephalomyocarditis virus internal ribosome entry 
                     site; IRES"
     misc_feature    729..1302
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     regulatory      1326..1334
                     /regulatory_class="ribosome_binding_site"
                     /note="Kozak site"
     regulatory      1329..1338
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1335..2420
                     /codon_start=1
                     /product="BirA"
                     /label=BirA
                     /note="holocarboxylase synthetase; secreted variant of 
                     Escherichia coli biotin ligase with an amino-terminal 
                     extension consisting of the bovine prolactin signal peptide
                     and a Flag epitope tag"
                     /protein_id="ABW71926.1"
                     /translation="MDSKGSSQKGSRLLLLLVVSNLLLCQGVVSDYKDDDDKLKMKDNT
                     VPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPI
                     QLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRRGRKW
                     FSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDR
                     KLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAM
                     LIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLL
                     EQDGIIKPWMGGEISLRSAEK"
     sig_peptide     1335..1424
                     /note="bovine prolactin signal peptide"
     misc_feature    1425..1448
                     /label=Region: Flag epitope tag
                     /note="Region: Flag epitope tag"
     CDS             1425..1448
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1452..2417
                     /note="native coding sequence of E. coli BirA; Region: 
                     biotin ligase"
     CDS             1455..2420
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     regulatory      2470..3637
                     /regulatory_class="polyA_signal_sequence"
     intron          2485..3057
                     /label=-beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     polyA_site      3254..3309
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     rep_origin      3839..4482
                     /label=Col E1 origin of replication
                     /note="Col E1 origin of replication"
     rep_origin      complement(3871..4459)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4630..5490)
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=beta-lactamase
                     /protein_id="ABW71927.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5491..5595)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5700..6029
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     regulatory      5713..6121
                     /regulatory_class="promoter"
     rep_origin      5880..6015
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6122..6721
                     /codon_start=1
                     /product="puromycin N-acetyltransferase"
                     /label=puromycin N-acetyltransferase
                     /protein_id="ABW71928.1"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    6951..7032
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"