LOCUS       Exported                6980 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pCW-LIC, complete sequence.
ACCESSION   EF460848
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6980)
  AUTHORS   Loppnau P, Kozieradzki I, Arrowsmith C.
  TITLE     Expression vectors for high-throughput In-Fusion cloning
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6980)
  AUTHORS   Loppnau P, Kozieradzki I, Arrowsmith C.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-FEB-2007) Structural Genomics Consortium, University 
            of Toronto, 100 College Street, Toronto, ON M5G1L5, Canada
REFERENCE   3  (bases 1 to 6980)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6980
                     /organism="Expression vector pCW-LIC"
                     /mol_type="other DNA"
                     /label=derived from pCWori+
                     /note="derived from pCWori+"
                     /db_xref="taxon:435750"
     rep_origin      12..467
                     /label=phage f1 replication origin
                     /note="phage f1 replication origin"
     rep_origin      12..440
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        492..596
                     /gene="bla"
                     /label=AmpR promoter
     CDS             597..1457
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /note="ampicillin resistance protein"
                     /protein_id="ABP57195.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            597..1457
                     /gene="bla"
                     /label=bla
     rep_origin      1628..2216
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      2215
                     /label=colE1 from pBR322
                     /note="colE1 from pBR322"
     misc_feature    2402..2544
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2665..3126)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVLPRAFR"
     primer_bind     3090..3106
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3122..3138)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3146..3162
                     /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)."
     promoter        complement(3170..3200)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3215..3236
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(3249..4331)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /protein_id="ABP57196.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(3249..4331)
                     /gene="lacI"
                     /label=lacI
     promoter        complement(4332..4409)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     promoter        4497..4527
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     regulatory      4521..4527
                     /regulatory_class="minus_10_signal"
                     /note="derived from lacUV5 promoter"
     protein_bind    4533..4553
                     /label=lac repressor protein binding site
                     /bound_moiety="lac repressor protein"
     protein_bind    4535..4551
                     /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)."
     regulatory      4606..4634
                     /regulatory_class="promoter"
                     /note="Ptac"
     promoter        4606..4634
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     regulatory      4606..4611
                     /regulatory_class="minus_35_signal"
                     /note="derived from trp promoter"
     regulatory      4628..4634
                     /regulatory_class="minus_10_signal"
                     /note="derived from lacUV5 promoter"
     protein_bind    4640..4660
                     /label=lac repressor protein binding site
                     /bound_moiety="lac repressor protein"
     protein_bind    4642..4658
                     /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)."
     regulatory      4701..4729
                     /regulatory_class="promoter"
                     /note="Ptac"
     promoter        4701..4729
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     regulatory      4701..4706
                     /regulatory_class="minus_35_signal"
                     /note="derived from trp promoter"
     regulatory      4723..4729
                     /regulatory_class="minus_10_signal"
                     /note="derived from lacUV5 promoter"
     protein_bind    4735..4755
                     /label=lac repressor protein binding site
                     /bound_moiety="lac repressor protein"
     protein_bind    4737..4753
                     /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    4784..4798
                     /note="5-cloning sequence; add to upstream PCR primer"
     protein_bind    4826..4859
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        4874..5319
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             5320..6741
                     /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 media containing 5% sucrose"
                     /protein_id="ABP57197.1"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     gene            5320..6741
                     /gene="sacB"
                     /label=sacB
     misc_feature    6814..6828
                     /note="3-cloning sequence; add complement to downstream 
                     PCR primer"
     regulatory      6862..6889
                     /regulatory_class="terminator"
                     /note="trp operon transcription termination sequence"