LOCUS       Exported                5189 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pAT224, complete sequence.
ACCESSION   AY327139
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5189)
  AUTHORS   Binz HK, Amstutz P, Kohl A, Stumpp MT, Briand C, Forrer P, Grutter 
            MG, Pluckthun A.
  TITLE     High-affinity binders selected from designed ankyrin repeat protein 
            libraries
  JOURNAL   Nat. Biotechnol. 22 (5), 575-582 (2004)
  PUBMED    15097997
REFERENCE   2  (bases 1 to 5189)
  AUTHORS   Stumpp MT, Forrer P, Binz KH, Amstutz P, Zahnd C, Pluckthun A.
  TITLE     Direct Submission
  JOURNAL   Submitted (20-JUN-2003) Biochemistry, University of Zurich, 
            Winterthurerstrasse 190, Zurich 8057, Switzerland
REFERENCE   3  (bases 1 to 5189)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5189
                     /organism="Expression vector pAT224"
                     /mol_type="genomic DNA"
                     /note="for the production of MBP fusion proteins that can 
                     be biotinylated"
                     /db_xref="taxon:248464"
     regulatory      7..87
                     /regulatory_class="promoter"
                     /note="T5"
     promoter        10..54
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             115..1338
                     /codon_start=1
                     /product="MBPfusion"
                     /label=MBPfusion
                     /note="Avi-tag_MBP_mcs_His-tag fusion protein; designed to 
                     increase ability to solubilize difficult proteins and 
                     improve expression yield"
                     /protein_id="AAQ93661.1"
                     /translation="MAGLNDIFEAQKIEWHEGSMGKTEEGKLVIWINGDKGYNGLAEVG
                     KKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDK
                     AFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAK
                     GKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKN
                     KHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVG
                     VLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAA
                     TMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGSGGTPGRPAAQ
                     ASHHHHHH"
     misc_feature    115..171
                     /label=Avi-tag
                     /note="Avi-tag"
     CDS             121..165
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     misc_feature    172..1275
                     /label=Escherichia coli maltose binding protein
                     /note="Escherichia coli maltose binding protein"
     CDS             172..1275
                     /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="MGKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKL
                     EEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGK
                     LIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLI
                     AADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKG
                     ETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEF
                     LENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSA
                     FWYAVRTAVINAASGRQTVDEALKDAQT"
     misc_feature    1276..1317
                     /label=multiple cloning site
                     /note="multiple cloning site"
     misc_feature    1318..1338
                     /label=6*His-tag
                     /note="6*His-tag"
     CDS             1318..1335
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1352..1446
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      1553..1639
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1699..2790)
                     /codon_start=1
                     /product="LacIq"
                     /label=LacIq
                     /protein_id="AAQ93659.1"
                     /translation="MVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAM
                     AELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVER
                     SGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD
                     WSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTED
                     SSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTA
                     SPRALADSLMQLARQVSRLESGQ"
     CDS             complement(1699..2781)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(2782..2859)
                     /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."
     misc_feature    3039..3179
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      3312..3968
                     /label=ColE1
                     /note="ColE1"
     rep_origin      complement(3365..3953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4124..4984)
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=beta-lactamase
                     /note="ampicillin resistance"
                     /protein_id="AAQ93660.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQAAMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4985..5089)
                     /gene="bla"
                     /label=AmpR promoter