LOCUS       Exported                5552 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pKLD66nCBP, complete sequence.
ACCESSION   KT020828
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5552)
  AUTHORS   St Maurice M.
  TITLE     The absence of substrate channeling in the biotin-dependent enzyme 
            urea amidolyase
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5552)
  AUTHORS   St Maurice M.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-JUN-2015) Biological Sciences, Marquette University, 
            530 N 15th Street, Milwaukee, WI 53233, USA
REFERENCE   3  (bases 1 to 5552)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5552
                     /organism="Expression vector pKLD66nCBP"
                     /mol_type="other DNA"
                     /db_xref="taxon:1748544"
     promoter        368..386
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    387..411
                     /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             426..448
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     misc_feature    456..536
                     /note="calmodulin binding peptide from muscle myosin light 
                     chain kinase"
     CDS             459..536
                     /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"
     misc_feature    576..596
                     /note="cleavage site for Tobacco Etch Virus (TEV); protease
                     cleavage site; allows site-specific proteolytic cleavage of
                     N-terminal CBP tag"
     CDS             576..596
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             647..664
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      731..778
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      815..1270
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        1297..1401
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1402..2262
                     /codon_start=1
                     /product="beta lactamase"
                     /EC_number="3.5.2.6"
                     /label=beta lactamase
                     /note="penicillin resistance"
                     /protein_id="ALM30214.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2433..3021
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3207..3349
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(3451..3642)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4451..5533)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="lactose operon transcriptional repressor"
                     /protein_id="ALM30215.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(4451..5533)
                     /gene="lacI"
                     /label=lacI
     promoter        complement(5534..59)
                     /gene="lacI"
                     /label=lacI promoter