LOCUS       Exported                6816 bp ds-DNA    circular SYN 20-3-2014
DEFINITION  Baculovirus vector encoding an N-terminal 6xHis-TEV cassette and 
            ampicillin resistance plus a SacB negative selection marker, for 
            purification of recombinant proteins.
ACCESSION   EF199842
VERSION     .
KEYWORDS    pFB-LIC-Bse
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6816)
  AUTHORS   Savitsky P, Bray J, Cooper CD, Marsden BD, Mahajan P, Burgess-Brown 
            NA, Gileadi O.
  TITLE     High-throughput production of human proteins for crystallization: 
            the SGC experience.
  JOURNAL   J. Struct. Biol. 2010;172:3-13.
  PUBMED    20541610
REFERENCE   2  (bases 1 to 6816)
  AUTHORS   Structural Genomics Consortium
  TITLE     Direct Submission
COMMENT     For ligation-independent cloning (LIC), linearize with BseRI to 
            remove the SacB cassette, and treat with T4 DNA polymerase plus 
            dGTP.
FEATURES             Location/Qualifiers
     source          1..6816
                     /organism="synthetic DNA construct"
                     /lab_host="Insect Cells"
                     /mol_type="other DNA"
     promoter        1..92
                     /gene="polh from Autographa californica"
                     /note="polyhedrin promoter"
                     /note="promoter for the baculovirus polyhedrin gene"
     CDS             148..150
                     /codon_start=1
                     /product="start codon"
                     /note="ATG"
                     /translation="M"
     CDS             154..171
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             196..216
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /note="TEV site"
                     /translation="ENLYFQS"
     protein_bind    246..279
                     /bound_moiety="Cre recombinase"
                     /note="loxP"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        294..739
                     /gene="sacR"
                     /note="sacB promoter"
                     /note="sacB promoter and control region"
     CDS             740..2161
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /note="SacB"
                     /note="negative selection marker"
                     /protein_id="
                     "
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     polyA_signal    2403..2537
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     mobile_element  2566..2731
                     /mobile_element_type="transposon:Tn7"
                     /note="Tn7L"
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      2915..3370
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        3397..3501
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             3502..4362
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4533..5121
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     mobile_element  5424..5648
                     /mobile_element_type="transposon:Tn7"
                     /note="Tn7R"
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(5715..6248)
                     /codon_start=1
                     /gene="aacC1"
                     /allele="
                     "
                     /product="gentamycin acetyltransferase"
                     /note="GmR"
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(6437..6465)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /note="Pc promoter"
                     /note="class 1 integron promoter"