LOCUS       Exported                7834 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pSFV4, complete sequence.
ACCESSION   KC164372
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7834)
  AUTHORS   Loppnau PA, Graslund S, Arrowsmith CH.
  TITLE     A pipeline to generate recombinant antibodies to human target 
            proteins involved in epigenetic mechanisms
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7834)
  AUTHORS   Loppnau PA, Graslund S, Arrowsmith CH.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-NOV-2012) Structural Genomics Consortium, University 
            of Toronto, MaRS Centre, South tower, 101 College St, Room 700, 
            Toronto, Ontario M5G 1L7, Canada
REFERENCE   3  (bases 1 to 7834)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7834
                     /organism="Expression vector pSFV4"
                     /mol_type="other DNA"
                     /db_xref="taxon:1292101"
     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="AGF34251.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            597..1457
                     /gene="bla"
                     /label=bla
     misc_feature    1312..1317
                     /gene="bla"
                     /label=Bsa1 site removed
                     /note="Bsa1 site removed"
     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="AGF34253.1"
                     /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    4796..4861
                     /note="corresponds to N-terminal pelB signal peptide 
                     sequence"
     misc_feature    4862..5127
                     /note="partial sequence of FAB light chain constant region"
     misc_feature    5113..5127
                     /label=5-In-Fusion cloning site
                     /note="5-In-Fusion cloning site"
     protein_bind    5143..5176
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        5191..5636
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             5637..7058
                     /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="AGF34252.1"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     gene            5637..7058
                     /gene="sacB"
                     /label=sacB
     misc_feature    7120..7418
                     /note="partial coding sequence of FAB heavy chain constant 
                     region"
     misc_feature    7120..7134
                     /label=3-In-Fusion cloning site
                     /note="3-In-Fusion cloning site"
     misc_feature    7419..7463
                     /label=C-terminal of FAB heavy chain with AviTag addition
                     /note="C-terminal of FAB heavy chain with AviTag addition"
     misc_feature    7464..7529
                     /note="C-terminal AviTag, in-vivo biotinylation signal 
                     sequence"
     CDS             7485..7529
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     misc_feature    7543..7587
                     /note="alternate C-terminal of FAB heavy chain without tag 
                     addition"
     misc_feature    7606..7644
                     /note="alternate C-terminal of FAB heavy chain with hexa 
                     histidine-tag"
     misc_feature    7645..7671
                     /label=C-terminal hexa histidine-tag
                     /note="C-terminal hexa histidine-tag"
     CDS             7654..7671
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      7716..7743
                     /regulatory_class="terminator"
                     /note="trp operon transcription termination sequence"