LOCUS       Exported                5825 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pCALnFLAG, complete sequence.
ACCESSION   AF087042
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5825)
  AUTHORS   Felts K, Grafsky AJ.
  TITLE     pCALnFLAG
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5825)
  AUTHORS   Felts K, Grafsky AJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (26-AUG-1998) Technical Services, Stratagene, 11011 N. 
            Torrey Pines Rd., La Jolla, CA 92037, USA
REFERENCE   3  (bases 1 to 5825)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5825
                     /organism="Cloning vector pCALnFLAG"
                     /lab_host="Escherichia coli K12"
                     /mol_type="genomic DNA"
                     /note="synthetic plasmid cloned and maintained in 
                     Escherichia coli"
                     /db_xref="taxon:81848"
     promoter        10..38
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      213..260
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     regulatory      complement(213..255)
                     /regulatory_class="terminator"
                     /note="T7 terminator"
     CDS             complement(323..508)
                     /codon_start=1
                     /product="calmodulin-binding peptide-FLAG epitope fusion 
                     protein"
                     /label=calmodulin-binding peptide-FLAG epitope fusion
                     /protein_id="AAC71016.1"
                     /translation="MKRRWKKNFIAVSAANRFKKISSSGALLVPRGSDYKDDDDKGRGS
                     EFSSRVLFHGSTRAQA"
     misc_feature    complement(386..409)
                     /note="FLAG epitope; other site"
     CDS             complement(386..409)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    complement(386..400)
                     /note="enterokinase target; other site"
     CDS             complement(410..427)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     misc_feature    complement(413..427)
                     /note="thrombin target; other site"
     misc_feature    complement(428..508)
                     /note="calmodulin-binding peptide; CBP; other site"
     CDS             complement(428..505)
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     RBS             516..538
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     regulatory      complement(553..596)
                     /regulatory_class="promoter"
                     /note="T7 lac"
     protein_bind    553..577
                     /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(578..596)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        905..982
                     /gene="lacI"
                     /label=lacI promoter
     CDS             974..2065
                     /codon_start=1
                     /product="lacI"
                     /label=lacI
                     /protein_id="AAC71018.1"
                     /translation="MVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAM
                     AELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVER
                     SGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD
                     WSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTED
                     SSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTA
                     SPRALADSLMQLARQVSRLESGQ"
     CDS             983..2065
                     /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"
     CDS             3377..3568
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    3670..3812
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3816..4756)
                     /direction=LEFT
                     /label=ColE1 origin of replication
                     /note="ColE1 origin of replication"
     rep_origin      complement(3998..4586)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4757..5617)
                     /codon_start=1
                     /product="beta-lactamase"
                     /function="ampicillin resistance"
                     /label=beta-lactamase
                     /protein_id="AAC71017.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVDAS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5618..5722)
                     /gene="bla"
                     /label=AmpR promoter