LOCUS       Exported                6831 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pBIG-5b, complete sequence.
ACCESSION   KM042177
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6831)
  AUTHORS   Kulman JD.
  TITLE     Mammalian expression vector for doxycycline-regulated production of 
            enzymatically biotinylated proteins
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6831)
  AUTHORS   Kulman JD.
  TITLE     Direct Submission
  JOURNAL   Submitted (21-JUN-2014) Puget Sound Blood Center Research Institute,
            Puget Sound Blood Center, 1551 Eastlake Ave E #100, Seattle, WA 
            98102, USA
REFERENCE   3  (bases 1 to 6831)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6831
                     /organism="Expression vector pBIG-5b"
                     /mol_type="other DNA"
                     /db_xref="taxon:1547497"
     CDS             complement(26..1030)
                     /codon_start=1
                     /product="FLAG-BirA"
                     /label=FLAG-BirA
                     /note="Escherichia coli biotin ligase; holocarboxylase 
                     synthetase; contains amino-terminal FLAG-tag"
                     /protein_id="AIR07418.1"
                     /translation="MDYKDDDDKSPRSMKDNTVPLKLIALLANGEFHSGEQLGETLGMS
                     RAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTN
                     QYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLS
                     LVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINM
                     AMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKL
                     DNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             complement(26..991)
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             complement(1004..1027)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1027..1036
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     regulatory      1051..1607
                     /regulatory_class="promoter"
                     /note="bidirectional Tet-responsive promoter"
     protein_bind    1175..1445
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        1478..1515
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    1654..1686
                     /label=multiple cloning site
                     /note="multiple cloning site"
     CDS             1693..1734
                     /codon_start=1
                     /product="biotin acceptor peptide"
                     /label=biotin acceptor peptide
                     /note="contains lysine residue to which d-biotin is 
                     enzymatically conjugated by an isopeptide linkage"
                     /protein_id="AIR07421.1"
                     /translation="MNDIFEAQKIEWH"
     misc_feature    1757..2337
                     /note="encephalomyocarditis virus internal ribosome entry 
                     site; IRES"
     misc_feature    1764..2337
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     regulatory      2399..2408
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2405..3124
                     /codon_start=1
                     /product="enhanced green fluorescent protein"
                     /label=enhanced green fluorescent protein
                     /protein_id="AIR07419.1"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    3141..4308
                     /label=beta-globin poly A signal region
                     /note="beta-globin poly A signal region"
     intron          3155..3727
                     /label=-beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     polyA_site      3924..3979
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     rep_origin      4509..5152
                     /label=Col E1 origin of replication
                     /note="Col E1 origin of replication"
     rep_origin      complement(4541..5129)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5300..6160)
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=beta-lactamase
                     /note="confers resistance to ampicillin"
                     /protein_id="AIR07420.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6161..6265)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    complement(6374..6825)
                     /label=SV40 poly A signal region
                     /note="SV40 poly A signal region"
     polyA_signal    6696..6830
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"