LOCUS       Exported                6844 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Site-specific excision vector pCRE5, complete sequence.
ACCESSION   EU215440
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6844)
  AUTHORS   Choi KH, Mima T, Casart Y, Rholl D, Kumar A, Beacham IR, Schweizer 
            HP.
  TITLE     Genetic tools for select-agent-compliant manipulation of 
            Burkholderia pseudomallei
  JOURNAL   Appl. Environ. Microbiol. 74 (4), 1064-1075 (2008)
  PUBMED    18156318
REFERENCE   2  (bases 1 to 6844)
  AUTHORS   Choi K-H., Schweizer HP.
  TITLE     Genetic tools for Pseudomonas and its related bacteria
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 6844)
  AUTHORS   Choi K-H., Schweizer HP.
  TITLE     Direct Submission
  JOURNAL   Submitted (21-SEP-2007) Microbiology, Immunology and Pathology, 
            Colorado State University, 1682 Campus Delivery, Ft. Collins, CO 
            80523-1682, USA
REFERENCE   4  (bases 1 to 6844)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6844
                     /organism="Site-specific excision vector pCRE5"
                     /mol_type="other DNA"
                     /db_xref="taxon:477959"
     CDS             complement(34..867)
                     /codon_start=1
                     /gene="rep-ts"
                     /product="temperature-sensitive Rep"
                     /label=rep-ts
                     /note="temperature-sensitive replication protein"
                     /protein_id="ABW35369.1"
                     /translation="MASPPMVYKSNALVEAAYCLSVQEQRIVLASISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMGSTHAIRLYELLTQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKARKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     gene            complement(34..867)
                     /gene="rep-ts"
                     /label=rep-ts
     rep_origin      868..1272
                     /note="region containing broad-host-range origin of 
                     replication from pRO1600"
     rep_origin      881..1232
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from 
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     regulatory      1286..1333
                     /regulatory_class="promoter"
                     /note="ribosomal S12 promoter from B. thailandensis E264"
     CDS             1448..2263
                     /codon_start=1
                     /gene="neo"
                     /product="kanamycin resistance protein"
                     /label=neo
                     /protein_id="ABW35370.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            1448..2263
                     /gene="neo"
                     /label=neo
     CDS             complement(2412..3443)
                     /codon_start=1
                     /gene="cre"
                     /product="Cre protein"
                     /label=cre
                     /protein_id="ABW35371.1"
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     gene            complement(2412..3443)
                     /gene="cre"
                     /label=cre
     regulatory      complement(3444..3510)
                     /regulatory_class="promoter"
                     /note="Escherichia coli rhaB promoter"
     promoter        complement(3475..3593)
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             3739..4575
                     /codon_start=1
                     /gene="rhaS"
                     /product="RhaS"
                     /label=rhaS
                     /note="rhamnose operon subunit"
                     /protein_id="ABW35372.1"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     gene            3739..4575
                     /gene="rhaS"
                     /label=rhaS
     CDS             4559..5497
                     /codon_start=1
                     /gene="rhaR"
                     /product="RhaR"
                     /label=rhaR
                     /note="rhamnose operon subunit"
                     /protein_id="ABW35373.1"
                     /translation="MAFCNNANLLNVFVRHIANNQLRSLAEVATVAHQLKLLKDDFFAS
                     DQQAVAVADRYPQDVFAEHTHDFCELVIVWRGNGLHVLNDRPYRITRGDLFYIHADDKH
                     SYASVNDLVLQNIIYCPERLKLNLDWQGAIPGFNASAGQPHWRLGSMGMAQARQVIGQL
                     EHESSQHVPFANEMAELLFGQLVMLLNRHRYTSDSLPPTSSETLLDKLITRLAASLKSP
                     FALDKFCDEASCSERVLRQQFRQQTGMTINQYLRQVRVCHAQYLLQHSRLLISDISTEC
                     GFEDSNYFSVVFTRETGMTPSQWRHLNSQKD"
     gene            4559..5497
                     /gene="rhaR"
                     /label=rhaR
     CDS             4649..5497
                     /codon_start=1
                     /gene="E. coli rhaR"
                     /product="transcriptional activator of rhaR and rhaS"
                     /label=rhaR
                     /note="needed for rhamnose-dependent activation of the rhaB
                     promoter"
                     /translation="MAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
                     D"
     primer_bind     complement(5531..5547)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5555..5571
                     /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(5579..5609)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5624..5645
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    5883..6139
                     /label=oriT
                     /note="oriT"
     oriT            6029..6137
                     /note="incP origin of transfer"
     rep_origin      complement(6209..6797)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"