LOCUS       Exported                7170 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pET21a-LIC, complete sequence.
ACCESSION   EF456737
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7170)
  AUTHORS   Loppnau P, Kozieradzki I, Arrowsmith C.
  TITLE     Expression vectors for high-throughput In-Fusion cloning
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7170)
  AUTHORS   Loppnau P, Kozieradzki I, Arrowsmith C.
  TITLE     Direct Submission
  JOURNAL   Submitted (26-FEB-2007) Structural Genomics Consortium, University 
            of Toronto, 100 College Street, Toronto, ON M5G1L5, Canada
REFERENCE   3  (bases 1 to 7170)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7170
                     /organism="Expression vector pET21a-LIC"
                     /mol_type="other DNA"
                     /db_xref="taxon:433625"
     misc_feature    12..239
                     /note="partial phage f1 replication origin; inactivated"
     CDS             260..1120
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /note="ampicillin resistance protein"
                     /protein_id="ABO60931.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            260..1120
                     /gene="bla"
                     /label=bla
     rep_origin      1291..1879
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      1878
                     /label=ColE1 pBR322
                     /note="ColE1 pBR322"
     misc_feature    2065..2207
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2309..2500)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3309..4391)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="from Escherichia coli"
                     /protein_id="ABO60932.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(3309..4391)
                     /gene="lacI"
                     /label=lacI
     promoter        complement(4392..4469)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        4782..4800
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4782..4798
                     /regulatory_class="promoter"
                     /note="T7"
     protein_bind    4801..4825
                     /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)."
     RBS             4840..4862
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     misc_feature    4870..4937
                     /label=N-terminal tag
                     /note="N-terminal tag"
     misc_feature    4873..4890
                     /label=hexahistidine tag
                     /note="hexahistidine tag"
     CDS             4873..4890
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    4915..4935
                     /label=TEV protease cleavage site
                     /note="TEV protease cleavage site"
     CDS             4915..4935
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     misc_feature    4924..4937
                     /note="5-LIC sequence; add to upstream PCR primer"
     protein_bind    4965..4998
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        5013..5458
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             5459..6880
                     /codon_start=1
                     /gene="sacB"
                     /product="levansucrase"
                     /label=sacB
                     /note="Bacillus subtilis negative selection gene; converts 
                     sucrose to a toxic product; allows selection of recombinant
                     plasmids on plates containing 5% sucrose"
                     /protein_id="ABO60933.1"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     gene            5459..6880
                     /gene="sacB"
                     /label=sacB
     misc_feature    6953..6966
                     /note="3-LIC sequence; add complement to downstream PCR 
                     primer"
     CDS             7014..7031
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      7098..7145
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     regulatory      7099..7145
                     /regulatory_class="terminator"
                     /note="T7"