LOCUS       Exported                6777 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Ubiquitin protease vector pHUsp2-cc, complete sequence.
ACCESSION   AY751540
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6777)
  AUTHORS   Catanzariti AM, Soboleva TA, Jans DA, Board PG, Baker RT.
  TITLE     An efficient system for high-level expression and easy purification 
            of authentic recombinant proteins
  JOURNAL   Protein Sci. 13 (5), 1331-1339 (2004)
  PUBMED    15096636
REFERENCE   2  (bases 1 to 6777)
  AUTHORS   Catanzariti AM, Baker RT.
  TITLE     Direct Submission
  JOURNAL   Submitted (15-SEP-2004) John Curtin School of Medical Research, 
            Australian National University, Mills Road, Canberra, ACT 2601, 
            Australia
REFERENCE   3  (bases 1 to 6777)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6777
                     /organism="Ubiquitin protease vector pHUsp2-cc"
                     /mol_type="other DNA"
                     /label=based on pET15b (Novagen)
                     /note="based on pET15b (Novagen)"
                     /db_xref="taxon:296596"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      21..37
                     /regulatory_class="promoter"
                     /note="T7 RNA polymerase promoter"
     protein_bind    40..64
                     /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             79..101
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             108..1244
                     /codon_start=1
                     /gene="Usp2"
                     /product="poly-histidine-tagged Usp2-cc"
                     /label=Usp2
                     /note="deubiquitylating enzyme; catalytic core of mouse 
                     Usp2"
                     /protein_id="AAV27298.1"
                     /translation="MGSSHHHHHHSSGLVPRGSHMLEDPLLTKAKNSKSAQGLAGLRNL
                     GNTCFMNSILQCLSNTRELRDYCLQRLYMRDLGHTSSAHTALMEEFAKLIQTIWTSSPN
                     DVVSPSEFKTQIQRYAPRFMGYNQQDAQEFLRFLLDGLHNEVNRVAARPKASPETLDHL
                     PDEEKGRQMWRKYLEREDSRIGDLFVGQLKSSLTCTDCGYCSTVFDPFWDLSLPIAKRG
                     YPEVTLMDCMRLFTKEDILDGDEKPTCCRCRARKRCIKKFSVQRFPKILVLHLKRFSES
                     RIRTSKLTTFVNFPLRDLDLREFASENTNHAVYNLYAVSNHSGTTMGGHYTAYCRSPVT
                     GEWHTFNDSSVTPMSSSQVRTSDAYLLFYELASPPSRM"
     gene            108..1244
                     /gene="Usp2"
                     /label=Usp2
     CDS             120..137
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             147..164
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      1309..1356
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(1531..1559)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     promoter        1672..1776
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1777..2637
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2808..3396
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3582..3724
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(3826..4017)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5329..6411)
                     /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(6412..6489)
                     /gene="lacI"
                     /label=lacI promoter