LOCUS       40924_13390        6424 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Site-specific excision vector pCRE3, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6424)
  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 6424)
  AUTHORS   Choi K-H., Schweizer HP.
  TITLE     Genetic tools for Pseudomonas and its related bacteria
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 6424)
  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 6424)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 6424)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Appl. 
            Environ. Microbiol."; date: "2008"; volume: "74"; issue: "4"; pages:
            "1064-1075"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted 
            (21-SEP-2007) Microbiology, Immunology and Pathology, Colorado State
            University, 1682 Campus Delivery, Ft. Collins, CO 80523-1682, USA"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6424
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(86..457)
                     /codon_start=1
                     /label=BleoR
                     /note="antibiotic-binding protein"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        complement(476..523)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     rep_origin      complement(643..994)
                     /direction=LEFT
                     /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)"
     CDS             1008..1838
                     /codon_start=1
                     /label=pRO1600 Rep
                     /note="replication protein for the broad-host-range plasmid
                     pRO1600 from Pseudomonas aeruginosa"
                     /translation="VASPPMVYKSNALVEAAYCLSVQEQRIVLASISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMGSTHAIRLYELLTQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKARKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     CDS             complement(1910..2755)
                     /codon_start=1
                     /label=rhaR
                     /note="transcriptional activator of rhaR and rhaS"
                     /translation="VAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
                     D"
     CDS             complement(2832..3665)
                     /codon_start=1
                     /label=rhaS
                     /note="positive regulator of the rhaB promoter"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     promoter        3811..3929
                     /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             3961..4989
                     /codon_start=1
                     /label=Cre
                     /note="site-specific recombinase"
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     primer_bind     complement(5111..5127)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5135..5151)
                     /label=lac operator
                     /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(5159..5189)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5204..5225)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     oriT            5609..5717
                     /label=oriT
                     /note="incP origin of transfer"
     rep_origin      complement(5789..6377)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cggtcgagcg gccgccagtg tgatggataa ttcggcttat ctctgatgtt acattgcaca
       61 agataaaata atatcatcat gatcagtcct gctcctcggc cacgaagtgc acgcagttgc
      121 cggccgggtc gcgcagggcg aactcccgcc cccacggctg ctcgccgatc tcggtcatgg
      181 ccggcccgga ggcgtcccgg aagttcgtgg acacgacctc cgaccactcg gcgtacagct
      241 cgtccaggcc gcgcacccac acccaggcca gggtgttgtc cggcaccacc tggtcctgga
      301 ccgcgctgat gaacagggtc acgtcgtccc ggaccacacc ggcgaagtcg tcctccacga
      361 agtcccggga gaacccgagc cggtcggtcc agaactcgac cgctccggcg acgtcgcgcg
      421 cggtgagcac cggaacggca ctggtcaact tggccatggt ttagttcctc accttgtcgt
      481 attatactat gccgatatac tatgccgatg attaattgtc aacacgtgct gatcatgacc
      541 aaaatccctt aacgtgagtt acgcgtcgaa gccgaattat ctgcagaatt cggcttttcg
      601 tgaattcgcc gctggtgccg ctggttggac gccaagggtg aatccgcctc gataccctga
      661 ttactcgctt cctgcgccct ctcaggcggc gataggggac tggtaaaacg gggattgccc
      721 agacgcctcc cccgcccctt caggggcaca aatgcggccc caacggggcc acgtagtggt
      781 gcgttttttg cgtttccacc cttttcttcc ttttcccttt taaacctttt aggacgtcta
      841 caggccacgt aatccgtggc ctgtagagtt taaaaaggga cggatttgtt gccattaagg
      901 gacggatttg ttgttaagaa gggacggatt tgttgttgta aagggacgga tttgttgtat
      961 tgtgggacgc tgatacagtg tccccttata cacaaggaat gtcgaacgtg gcctcacccc
     1021 caatggttta caaaagcaat gccctggtcg aggccgcgta ttgcctcagt gttcaggaac
     1081 agcggatcgt tctggccagt attagccagg tgaagaggag cgagcctgtc accgatgaag
     1141 tgatgtattc agtgacggcg gaggacatag cgacgatggc gggtgtccct atcgaatctt
     1201 cctacaacca gctcaaagaa gcggccctgc gcctgaaacg gcgggaagtc cggttaaccc
     1261 aagagcccaa tggcaagggg aaaagaccga gtgtgatgat taccggctgg gtgcaaacaa
     1321 tcatctaccg ggagggtgag ggccgtgtag aactcaggtt caccaaagac atgctgccgt
     1381 atctgacgga actcaccaaa cagttcacca aatacgcctt ggctgacgtg gccaagatgg
     1441 gcagcaccca cgcgatcagg ctttacgagc tgctcacgca atgggacagc atcggccagc
     1501 gtgaaataga aattgaccag ctgcgaaagt ggtttcaact ggaaggccgg tatccctcga
     1561 tcaaggactt caagttgcga gtgcttgatc cagccgtgac gcagatcaac gagcacagcc
     1621 cgctacaggt ggagtgggcg cagcgaaaga ccgggcgcaa ggtcacacat ctgttgttca
     1681 gttttggacc gaagaagccc gccaaggcgg tgggtaaggc ccctgcgaag cgcaaggcca
     1741 ggaagatttc agatgctgag atcgcgaaac aggctcgccc tggtgagaca tgggaagcgg
     1801 cccgcgctcg actaacccag atgccgctgg atctggccta gaggccaagc cgaattccag
     1861 cacactggcg gccgttacta gtggatccga gctcgatcat atgcatttaa tctttctgcg
     1921 aattgagatg acgccactgg ctgggcgtca tcccggtttc ccgggtaaac accaccgaaa
     1981 aatagttact atcttcaaag ccacattcgg tcgaaatatc actgattaac aggcggctat
     2041 gctggagaag atattgcgca tgacacactc tgacctgtcg cagatattga ttgatggtca
     2101 ttccagtctg ctggcgaaat tgctgacgca aaacgcgctc actgcacgat gcctcatcac
     2161 aaaatttatc cagcgcaaag ggacttttca ggctagccgc cagccgggta atcagcttat
     2221 ccagcaacgt ttcgctggat gttggcggca acgaatcact ggtgtaacga tggcgattca
     2281 gcaacatcac caactgcccg aacagcaact cagccatttc gttagcaaac ggcacatgct
     2341 gactactttc atgctcaagc tgaccgataa cctgccgcgc ctgcgccatc cccatgctac
     2401 ctaagcgcca gtgtggttgc cctgcgctgg cgttaaatcc cggaatcgcc ccctgccagt
     2461 caagattcag cttcagacgc tccgggcaat aaataatatt ctgcaaaacc agatcgttaa
     2521 cggaagcgta ggagtgttta tcgtcagcat gaatgtaaaa gagatcgcca cgggtaatgc
     2581 gataagggcg atcgttgagt acatgcaggc cattaccgcg ccagacaatc accagctcac
     2641 aaaaatcatg tgtatgttca gcaaagacat cttgcggata acggtcagcc acagcgactg
     2701 cctgctggtc gctggcaaaa aaatcatctt tgagaagttt taactgatgc gccaccgtgg
     2761 ctacctcggc cagagaacga agttgattat tcgcaatatg gcgtacaaat acgttgagaa
     2821 gattcgcgtt attgcagaaa gccatcccgt ccctggcgaa tatcacgcgg tgaccagtta
     2881 aactctcggc gaaaaagcgt cgaaaagtgg ttactgtcgc tgaatccaca gcgataggcg
     2941 atgtcagtaa cgctggcctc gctgtggcgt agcagatgtc gggctttcat cagtcgcagg
     3001 cggttcaggt atcgctgagg cgtcagtccc gtttgctgct taagctgccg atgtagcgta
     3061 cgcagtgaaa gagaaaattg atccgccacg gcatcccaat tcacctcatc ggcaaaatgg
     3121 tcctccagcc aggccagaag caagttgaga cgtgatgcgc tgttttccag gttctcctgc
     3181 aaactgcttt tacgcagcaa gagcagtaat tgcataaaca agatctcgcg actggcggtc
     3241 gagggtaaat cattttcccc ttcctgctgt tccatctgtg caaccagctg tcgcacctgc
     3301 tgcaatacgc tgtggttaac gcgccagtga gacggatact gcccatccag ctcttgtggc
     3361 agcaactgat tcagcccggc gagaaactga aatcgatccg gcgagcgata cagcacattg
     3421 gtcagacaca gattatcggt atgttcatac agatgccgat catgatcgcg tacgaaacag
     3481 accgtgccac cggtgatggt atagggctgc ccattaaaca catgaatacc cgtgccatgt
     3541 tcgacaatca caatttcatg aaaatcatga tgatgttcag gaaaatccgc ctgcgggagc
     3601 cggggttcta tcgccacgga cgcgttacca gacggaaaaa aatccacact atgtaatacg
     3661 gtcatactgg cctcctgatg tcgtcaacac ggcgaaatag taatcacgag gtcaggttct
     3721 taccttaaat tttcgacgga aaaccacgta aaaaacgtcg atttttcaag atacagcgtg
     3781 aattttcagg aaatgcggtg agcatcacat caccacaatt cagcaaattg tgaacatcat
     3841 cacgttcatc tttccctggt tgccaatggc ccattttcct gtcagtaacg agaaggtcgc
     3901 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag caccattatc tgagtgttaa
     3961 atgtccaatt tactgaccgt acaccaaaat ttgcctgcat taccggtcga tgcaacgagt
     4021 gatgaggttc gcaagaacct gatggacatg ttcagggatc gccaggcgtt ttctgagcat
     4081 acctggaaaa tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac
     4141 cggaaatggt ttcccgcaga acctgaagat gttcgcgatt atcttctata tcttcaggcg
     4201 cgcggtctgg cagtaaaaac tatccagcaa catttgggcc agctaaacat gcttcatcgt
     4261 cggtccgggc tgccacgacc aagtgacagc aatgctgttt cactggttat gcggcggatc
     4321 cgaaaagaaa acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact
     4381 gatttcgacc aggttcgttc actcatggaa aatagcgatc gctgccagga tatacgtaat
     4441 ctggcatttc tggggattgc ttataacacc ctgttacgta tagccgaaat tgccaggatc
     4501 agggttaaag atatctcacg tactgacggt gggagaatgt taatccatat tggcagaacg
     4561 aaaacgctgg ttagcaccgc aggtgtagag aaggcactta gcctgggggt aactaaactg
     4621 gtcgagcgat ggatttccgt ctctggtgta gctgatgatc cgaataacta cctgttttgc
     4681 cgggtcagaa aaaatggtgt tgccgcgcca tctgccacca gccagctatc aactcgcgcc
     4741 ctggaaggga tttttgaagc aactcatcga ttgatttacg gcgctaagga tgactctggt
     4801 cagagatacc tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg agatatggcc
     4861 cgcgctggag tttcaatacc ggagatcatg caagctggtg gctggaccaa tgtaaatatt
     4921 gtcatgaact atatccgtaa cctggatagt gaaacagggg caatggtgcg cctgctggaa
     4981 gatggcgatt agccattaac gcgtaaatga ttgctataat tagttgatag tcgacctgca
     5041 gatctatcac tagtgaattc gcggccgcct gcaggtcgac catatgggag agctcgaatt
     5101 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5161 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5221 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5281 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5341 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5401 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgggga
     5461 ttccttaagg tatactttcc gctgcataac cctgcttcgg ggtcattata gcgatttttt
     5521 cggtatatcc atcctttttc gcacgatata caggattttg ccaaagggtt cgtgtagact
     5581 ttccttggtg tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga
     5641 gcgggtgttc cttcttcact gtcccttatt cgcacctggc ggtgctcaac gggaatcctg
     5701 ctctgcgagg ctggccgata agctagctta tgtgagcaaa aggccagcaa aaggccagga
     5761 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5821 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5881 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5941 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6001 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6061 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6121 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6181 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     6241 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6301 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6361 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6421 acga
//
