LOCUS       Exported                6665 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pOPINM, complete sequence.
ACCESSION   EF372396
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6665)
  AUTHORS   Berrow NS, Alderton D, Sainsbury S, Nettleship J, Assenberg R, 
            Rahman N, Stuart DI, Owens RJ.
  TITLE     A versatile ligation-independent cloning method suitable for 
            high-throughput expression screening applications
  JOURNAL   Nucleic Acids Res. 35 (6), E45 (2007)
  PUBMED    17317681
REFERENCE   2  (bases 1 to 6665)
  AUTHORS   Berrow NS.
  TITLE     Direct Submission
  JOURNAL   Submitted (17-JAN-2007) Oxford Protein Production Facility, Oxford 
            University, Roosevelt Drive, Oxford, Oxfordshire OX3 7BN, U.K.
REFERENCE   3  (bases 1 to 6665)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6665
                     /organism="Expression vector pOPINM"
                     /mol_type="other DNA"
                     /label=based on pTriEx2 backbone
                     /note="based on pTriEx2 backbone"
                     /db_xref="taxon:425269"
     misc_feature    45..1011
                     /label=lef2/ORF603
                     /note="lef2/ORF603"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     regulatory      1079..1443
                     /regulatory_class="enhancer"
                     /note="CMV enhancer"
     enhancer        1140..1443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     regulatory      1449..1726
                     /regulatory_class="promoter"
                     /note="chicken beta-actin promoter"
     promoter        1449..1726
                     /label=chicken beta-actin promoter
     promoter        2150..2168
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2169..2193
                     /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)."
     misc_feature    2171..2191
                     /label=lac operator
                     /note="lac operator"
     misc_feature    2205..2318
                     /label=p10 promoter and 5 UTR
                     /note="p10 promoter and 5 UTR"
     promoter        2209..2318
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     regulatory      2318..2326
                     /regulatory_class="ribosome_binding_site"
     misc_feature    2333..3499
                     /label=MAHHHHHHSSG-MBP-3C tag
                     /note="MAHHHHHHSSG-MBP-3C tag"
     CDS             2339..2356
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2366..3466
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     misc_feature    3480..3494
                     /label=5 in-fusion site (after KpnI disgest)
                     /note="5 in-fusion site (after KpnI disgest)"
     misc_feature    3500..3838
                     /label=lacZ promoter and lacZ alpha peptide
                     /note="lacZ promoter and lacZ alpha peptide"
     protein_bind    3540..3561
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3576..3606
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3614..3630
                     /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)."
     primer_bind     3638..3654
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3650..3823
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     complement(3670..3686)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    3829..3846
                     /label=3 in-fusion site (after HindIII digest)
                     /note="3 in-fusion site (after HindIII digest)"
     CDS             3853..3870
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      3922..4164
                     /regulatory_class="polyA_signal_sequence"
                     /note="rabbit beta-globin"
     polyA_site      4025..4080
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     regulatory      4168..4215
                     /regulatory_class="terminator"
                     /note="T7 terminator"
     terminator      4168..4215
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_feature    4228..4933
                     /label=ORF1629
                     /note="ORF1629"
     misc_recomb     4228..4933
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3UTR           4316..4688
                     /label=polyhedrin 3 UTR
                     /note="3 UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4345..4933)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    4942..4975
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     rep_origin      complement(5143..5730)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      5144
                     /label=pUC origin of replication
                     /note="pUC origin of replication"
     CDS             complement(5743..6603)
                     /codon_start=1
                     /gene="ampR"
                     /product="beta-lactamase"
                     /label=ampR
                     /protein_id="ABN80181.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(5743..6603)
                     /gene="ampR"
                     /label=ampR