LOCUS       Exported                4366 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pAT223, complete sequence.
ACCESSION   AY327138
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4366)
  AUTHORS   Amstutz P, Binz HK, Parizek P, Stumpp MT, Kohl A, Grutter MG, Forrer
            P, Pluckthun A.
  TITLE     Intracellular kinase inhibitors selected from combinatorial 
            libraries of designed ankyrin repeat proteins
  JOURNAL   J. Biol. Chem. 280 (26), 24715-24722 (2005)
  PUBMED    15851475
REFERENCE   2  (bases 1 to 4366)
  AUTHORS   Stumpp MT, Forrer P, Binz HK, Amstutz P, Zahnd C, Pluckthun A.
  TITLE     Designed ankyrin repeat proteins binding maltose binding protein
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 4366)
  AUTHORS   Stumpp MT, Forrer P, Binz HK, Amstutz P, Zahnd C, Pluckthun A.
  TITLE     Direct Submission
  JOURNAL   Submitted (20-JUN-2003) Biochemistry, University of Zurich, 
            Winterthurerstrasse 190, Zurich 8057, Switzerland
REFERENCE   4  (bases 1 to 4366)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4366
                     /organism="Expression vector pAT223"
                     /mol_type="genomic DNA"
                     /note="for the production of protein D fusion proteins that
                     can be biotinylated"
                     /db_xref="taxon:248463"
     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..519
                     /codon_start=1
                     /product="pDfusion"
                     /label=pDfusion
                     /note="Avi-tag_pD_His-tag_mcs fusion protein"
                     /protein_id="AAQ93658.1"
                     /translation="MAGLNDIFEAQKIEWHEGSMGTATAPGGLSAKAPAMTPLMLDTSS
                     RKLVAWDGTTDGAAVGILAVAADQTSTTLTFYKSGTFRYEDVLWPEAASDETKKRTAFA
                     GTAISIVGSGHHHHHHGSGGTPGRPAAKLN"
     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..456
                     /label=bacteriophage lambda protein D
                     /note="bacteriophage lambda protein D"
     misc_feature    457..474
                     /label=6*His-tag
                     /note="6*His-tag"
     CDS             457..474
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    475..519
                     /label=multiple cloning site
                     /note="multiple cloning site"
     terminator      529..623
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      730..816
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(876..1967)
                     /codon_start=1
                     /product="LacIq"
                     /label=LacIq
                     /protein_id="AAQ93656.1"
                     /translation="MVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAM
                     AELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVER
                     SGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD
                     WSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTED
                     SSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTA
                     SPRALADSLMQLARQVSRLESGQ"
     CDS             complement(876..1958)
                     /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(1959..2036)
                     /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    2216..2356
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      2489..3145
                     /label=ColE1
                     /note="ColE1"
     rep_origin      complement(2542..3130)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3301..4161)
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=beta-lactamase
                     /note="ampicillin resistance"
                     /protein_id="AAQ93657.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQAAMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4162..4266)
                     /gene="bla"
                     /label=AmpR promoter