LOCUS       40924_46528        5092 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gene replacement vector pwFRT-PCS12-gat-sacB, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5092)
  AUTHORS   Kang Y, Norris MH, Wilcox BA, Hoang T.
  TITLE     Knock-out and pull-out recombineering protocols for naturally 
            transformable Burkholderia thailandensis and Burkholderia 
            pseudomallei
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5092)
  AUTHORS   Kang Y, Norris MH, Wilcox BA, Hoang T.
  TITLE     Direct Submission
  JOURNAL   Submitted (09-JAN-2010) Molecular Biosciences and Bioengineering, 
            University of Hawaii at Manoa, 2538 McCarthy Mall, Snyder Hall 207, 
            Honolulu, HI 96822, USA
REFERENCE   3  (bases 1 to 5092)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5092)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (09-JAN-2010) Molecular Biosciences and Bioengineering, University 
            of Hawaii at Manoa, 2538 McCarthy Mall, Snyder Hall 207, Honolulu, 
            HI 96822, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5092
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        96..200
                     /label=AmpR promoter
     CDS             201..1058
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1232..1820
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2144..2174
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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)."
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_recomb     2289..2356
                     /note="FRT; flp recognition target"
     protein_bind    complement(2306..2353)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     regulatory      2393..2434
                     /note="PCS12; Burkholderia cenocepacia rpsL promoter"
                     /regulatory_class="promoter"
     RBS             2445..2467
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2503..2943
                     /codon_start=1
                     /gene="gat"
                     /product="glyphosate acetyl transferase"
                     /label=gat
                     /note="Gat; confers resistance to glyphosate"
                     /protein_id="ADD38967.1"
                     /translation="MIEVKPINAEDTYDLRHRVLRPNQPIEACMFESDLTRSAFHLGGF
                     YGGKLISVASFHQAEHSELQGKKQYQLRGVATLEGYREQKAGSSLVKHAEEILRKRGAD
                     MIWCNARTSASGYYRKLGFSEQGEVFDTPPVGPHILMYKRIT"
     gene            2503..2943
                     /gene="gat"
                     /label=gat
     CDS             3105..4457
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     terminator      complement(4474..4517)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     protein_bind    complement(4584..4631)
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     complement(4698..4714)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctt gcatgcctgc aggtcgactc taggatcccc gggtaccgag
     2281 ctcgaattag cttcaaaagc gctctgaagt tcctatactt tctagagaat aggaacttcg
     2341 gaataggaac ttcaagatcc cctgattccc tttgtcaaca gcaatggata tcagccgttg
     2401 acttagttgg tatttccgga atatcatgct gggttccgaa taattttgtt taactttaag
     2461 aaggagatat accatgggat cgagcttcga aaggacaagc atatgatcga ggtcaagccc
     2521 atcaacgccg aggacaccta cgacctgcgg catcgggtcc tccggcccaa ccagccgatc
     2581 gaggcgtgca tgttcgaaag cgatctcacg cggagcgcct tccacctggg cgggttctac
     2641 gggggcaagc tgatttcggt cgcttcgttc caccaggccg agcactcgga gcttcagggg
     2701 aagaagcagt accagctgcg cggtgtggcc accctggagg gctaccggga gcagaaggcc
     2761 gggtcgtcgc tggtcaagca cgccgaagag atcctgcgga agcggggggc cgacatgatc
     2821 tggtgcaacg cccggacctc ggcctcgggg tactaccgga agctggggtt cagcgagcag
     2881 ggggaggtct tcgacacccc gcccgtgggg ccccacatcc tgatgtacaa gcggatcacc
     2941 taactcgagt ttgtcaaaac ccgtccatat aattagcact cgctgcacag gaggaaacga
     3001 tgacgtactc caaacaagcc gtttcccgta gacacaaacg tttggccctg agcgccgccg
     3061 cgctcaccgc agccgcttgc atgtcggcgc acgcgcagag cgacggcgca actcaagcgt
     3121 ttgcgaaaga aacgaaccaa aagccatata aggaaacata cggcatttcc catattacac
     3181 gccatgatat gctgcaaatc cctgaacagc aaaaaaatga aaaatatcaa gtttctgaat
     3241 ttgattcgtc cacaattaaa aatatctctt ctgcaaaagg cctggacgtt tgggacagct
     3301 ggccattaca aaacgctgac ggcactgtcg caaactatca cggctaccac atcgtctttg
     3361 cattagccgg agatcctaaa aatgcggatg acacatcgat ttacatgttc tatcaaaaag
     3421 tcggcgaaac ttctattgac agctggaaaa acgctggccg cgtctttaaa gacagcgaca
     3481 aattcgatgc aaatgattct atcctaaaag accaaacaca agaatggtca ggttcagcca
     3541 catttacatc tgacggaaaa atccgtttat tctacactga tttctccggt aaacattacg
     3601 gcaaacaaac actgacaact gcacaagtta acgtatcagc atcagacagc tctttgaaca
     3661 tcaacggtgt agaggattat aaatcaatct ttgacggtga cggaaaaacg tatcaaaatg
     3721 tacagcagtt catcgatgaa ggcaactaca gctcaggcga caaccatacg ctgagagatc
     3781 ctcactacgt agaagataaa ggccacaaat acttagtatt tgaagcaaac actggaactg
     3841 aagatggcta ccaaggcgaa gaatctttat ttaacaaagc atactatggc aaaagcacat
     3901 cattcttccg tcaagaaagt caaaaacttc tgcaaagcga taaaaaacgc acggctgagt
     3961 tagcaaacgg cgctctcggt atgattgagc taaacgatga ttacacactg aaaaaagtga
     4021 tgaaaccgct gattgcatct aacacagtaa cagatgaaat tgaacgcgcg aacgtcttta
     4081 aaatgaacgg caaatggtac ctgttcactg actcccgcgg atcaaaaatg acgattgacg
     4141 gcattacgtc taacgatatt tacatgcttg gttatgtttc taattcttta actggcccat
     4201 acaagccgct gaacaaaact ggccttgtgt taaaaatgga tcttgatcct aacgatgtaa
     4261 cctttactta ctcacacttc gctgtacctc aagcgaaagg aaacaatgtc gtgattacaa
     4321 gctatatgac aaacagagga ttctacgcag acaaacaatc aacgtttgcg ccgagcttcc
     4381 tgctgaacat caaaggcaag aaaacatctg ttgtcaaaga cagcatcctt gaacaaggac
     4441 aattaacagt taacaaataa tagtagcttt caaataaaac gaaaggctca gtcgaaagac
     4501 tgggcctttc gttttaatct gagacgtcct cgagctaatt ccattgagta agtttttaag
     4561 cacatcagct tcaaaagcgc tctgaagttc ctatactttc tagagaatag gaacttcgga
     4621 ataggaactt caagatcccc aattcgagct cggtacccgg ggatcctaga gtcgacctgc
     4681 aggcatgcaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     4741 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     4801 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg
     4861 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
     4921 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     4981 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5041 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc ga
//
