LOCUS       Exported                6895 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Mammalian expression vector pNBio, complete sequence.
ACCESSION   DQ520290
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6895)
  AUTHORS   Kulman JD, Satake M, Harris JE.
  TITLE     A versatile system for site-specific enzymatic biotinylation and 
            regulated expression of proteins in cultured mammalian cells
  JOURNAL   Protein Expr. Purif. 52 (2), 320-328 (2007)
  PUBMED    17084093
REFERENCE   2  (bases 1 to 6895)
  AUTHORS   Kulman JD, Satake M, Harris JE.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-APR-2006) Biochemistry, University of Washington, N.E.
            Pacific St., Seattle, WA 98195, USA
REFERENCE   3  (bases 1 to 6895)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6895
                     /organism="Mammalian expression vector pNBio"
                     /mol_type="other DNA"
                     /db_xref="taxon:386594"
     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"
     regulatory      480..489
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     misc_feature    492..530
                     /label=biotin acceptor peptide (BioTag)-encoding
                     /note="biotin acceptor peptide (BioTag)-encoding"
     misc_feature    532..551
                     /note="multiple cloning site; MCS"
     misc_feature    645..1225
                     /note="encephalomyocarditis virus internal ribosome entry 
                     site; IRES"
     misc_feature    652..1225
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     gene            1270..2283
                     /gene="birA"
                     /label=birA
     regulatory      1270..1278
                     /regulatory_class="ribosome_binding_site"
                     /gene="birA"
                     /note="Kozak site"
     regulatory      1273..1282
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1279..2283
                     /codon_start=1
                     /gene="birA"
                     /product="biotin ligase"
                     /label=birA
                     /note="holocarboxylase synthetase"
                     /protein_id="ABF74574.1"
                     /translation="MDYKDDDDKSPRSMKDNTVPLKLIALLANGEFHSGEQLGETLGMS
                     RAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTN
                     QYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLS
                     LVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINM
                     AMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKL
                     DNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     misc_feature    1282..1305
                     /gene="birA"
                     /label=FLAG epitope tag-encoding
                     /note="FLAG epitope tag-encoding"
     CDS             1282..1305
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1318..2283
                     /gene="birA"
                     /label=Escherichia coli BirA (biotin ligase)
                     /note="Escherichia coli BirA (biotin ligase)"
     CDS             1318..2283
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     regulatory      2333..3500
                     /regulatory_class="polyA_signal_sequence"
                     /note="beta-globin poly-A signal"
     intron          2348..2920
                     /label=-beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     polyA_site      3117..3172
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     rep_origin      3702..4345
                     /label=Col E1 origin of replication
                     /note="Col E1 origin of replication"
     rep_origin      complement(3734..4322)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4493..5353)
                     /codon_start=1
                     /gene="Ampr"
                     /product="beta-lactamase"
                     /label=Ampr
                     /note="ampicillin resistance"
                     /protein_id="ABF74575.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(4493..5353)
                     /gene="Ampr"
                     /label=Ampr
     promoter        complement(5354..5458)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5563..5892
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     regulatory      5576..5984
                     /regulatory_class="promoter"
                     /note="SV40 promoter"
     rep_origin      5743..5878
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5985..6584
                     /codon_start=1
                     /product="puromycin N-acetyltransferase"
                     /label=puromycin N-acetyltransferase
                     /protein_id="ABF74576.1"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     regulatory      6524..6834
                     /regulatory_class="polyA_signal_sequence"
                     /note="SV40 poly-A signal"
     polyA_signal    6814..6895
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"