LOCUS       40924_32145        7353 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle/allelic-replacement vector pMQ75, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7353)
  AUTHORS   Shanks RM, Caiazza NC, Hinsa SM, Toutain CM, O'toole GA.
  TITLE     Saccharomyces cerevisiae-Based Molecular Tool Kit for Manipulation 
            of Genes from Gram-Negative Bacteria
  JOURNAL   Appl. Environ. Microbiol. 72 (7), 5027-5036 (2006)
  PUBMED    16820502
REFERENCE   2  (bases 1 to 7353)
  AUTHORS   Shanks RMQ., Caiazza NC, Hinsa SM, Toutain CM, O'Toole GA.
  TITLE     Direct Submission
  JOURNAL   Submitted (15-SEP-2005) Microbiology and Immunology, Dartmouth 
            Medical School, Vail Building Room 505, Hanover, NH 03755, USA
REFERENCE   3  (bases 1 to 7353)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7353)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Appl. 
            Environ. Microbiol."; date: "2006"; volume: "72"; issue: "7"; pages:
            "5027-5036"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (15-SEP-2005) Microbiology and Immunology, Dartmouth Medical School,
            Vail Building Room 505, Hanover, NH 03755, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7353
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(212..1630)
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        complement(1631..2076)
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     oriT            complement(2450..2559)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     terminator      complement(2980..3007)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(3099..3185)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     primer_bind     3535..3551
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    complement(3555..3611)
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3621..3637)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3645..3661)
                     /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(3669..3699)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3714..3735)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      3862..4406
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     CDS             complement(4766..5296)
                     /codon_start=1
                     /label=GmR
                     /note="gentamycin acetyltransferase"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(5485..5513)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        complement(5531..5635)
                     /label=AmpR promoter
     CDS             complement(5735..6535)
                     /codon_start=1
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(6536..6756)
                     /label=URA3 promoter
     misc_feature    6784..7287
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataatcg gcattttctt ttgcgttttt atttgttaac tgttaattgt ccttgttcaa
      241 ggatgctgtc tttgacaaca gatgttttct tgcctttgat gttcagcagg aagctaggcg
      301 caaacgttga ttgtttgtct gcgtagaatc ctctgtttgt catatagctt gtaatcacga
      361 cattgtttcc tttcgcttga ggtacagcga agtgtgagta agtaaaggtt acatcgttag
      421 gatcaagatc catttttaac acaaggccag ttttgttcag cggcttgtat gggccagtta
      481 aagaattaga aacataacca agcatgtaaa tatcgttaga cgtaatgccg tcaatcgtca
      541 tttttgatcc gcgggagtca gtgaacagat accatttgcc gttcatttta aagacgttcg
      601 cgcgttcaat ttcatctgtt actgtgttag atgcaatcag cggtttcatc acttttttca
      661 gtgtgtaatc atcgtttagc tcaatcatac cgagagcgcc gtttgctaac tcagccgtgc
      721 gttttttatc gctttgcaga agtttttgac tttcttgacg gaagaatgat gtgcttttgc
      781 catagtatgc tttgttaaat aaagattctt cgccttggta gccatcttca gttccagtgt
      841 ttgcttcaaa tactaagtat ttgtggcctt tatcttctac gtagtgagga tctctcagcg
      901 tatggttgtc gcctgagctg tagttgcctt catcgatgaa ctgctgtaca ttttgatacg
      961 tttttccgtc accgtcaaag attgatttat aatcctctac accgttgatg ttcaaagagc
     1021 tgtctgatgc tgatacgtta acttgtgcag ttgtcagtgt ttgtttgccg taatgtttac
     1081 cggagaaatc agtgtagaat aaacggattt ttccgtcaga tgtaaatgtg gctgaacctg
     1141 accattcttg tgtttggtct tttaggatag aatcatttgc atcgaatttg tcgctgtctt
     1201 taaagacgcg gccagcgttt ttccagctgt caatagaagt ttcgccgact ttttgataga
     1261 acatgtaaat cgatgtgtca tccgcatttt taggatctcc ggctaatgca aagacgatgt
     1321 ggtagccgtg atagtttgcg acagtgccgt cagcgttttg taatggccag ctgtcccaaa
     1381 cgtccaggcc ttttgcagaa gagatatttt taattgtgga cgaatcgaac tcaggaactt
     1441 gatatttttc atttttttgc tgttcaggga tttgcagcat atcatggcgt gtaatatggg
     1501 aaatgccgta tgtttcctta tatggctttt ggttcgtttc tttcgcaaac gcttgagttg
     1561 cgcctcctgc cagcagtgcg gtagtaaagg ttaatactgt tgcttgtttt gcaaactttt
     1621 tgatgttcat cgttcatgtc tcctttttta tgtactgtgt tagcggtctg cttcttccag
     1681 ccctcctgtt tgaagatggc aagttagtta cgcacaataa aaaaagacct aaaatatgta
     1741 aggggtgacg ccaaagtata cactttgccc tttacacatt ttaggtcttg cctgctttat
     1801 cagtaacaaa cccgcgcgat ttacttttcg acctcattct attagactct cgtttggatt
     1861 gcaactggtc tattttcctc ttttgtttga tagaaaatca taaaaggatt tgcagactac
     1921 gggcctaaag aactaaaaaa tctatctgtt tcttttcatt ctctgtattt tttatagttt
     1981 ctgttgcatg ggcataaagt tgccttttta atcacaattc agaaaatatc ataatatctc
     2041 atttcactaa ataatagtga acggcaggta tatgtgatgg gttaaaaagg atcgatcctc
     2101 tagctagagt cgatcttcgc cagcagggcg aggatcgtgg catcaccgaa ccgcgccgtg
     2161 cgcgggtcgt cggtgagcca gagtttcagc aggccgccca ggcggcccag gtcgccattg
     2221 atgcgggcca gctcgcggac gtgctcatag tccacgacgc ccgtgatttt gtagccctgg
     2281 ccgacggcca gcaggtaggc cgacaggctc atgccggccg ccgccgcctt ttcctcaatc
     2341 gctcttcgtt cgtctggaag gcagtacacc ttgataggtg ggctgccctt cctggttggc
     2401 ttggtttcat cagccatccg cttgccctca tctgttacgc cggcggtagc cggccagcct
     2461 cgcagagcag gattcccgtt gagcaccgcc aggtgcgaat aagggacagt gaagaaggaa
     2521 cacccgctcg cgggtgggcc tacttcacct atcctgcccg gctgacgccg ttggatacac
     2581 caaggaaagt ctacacgaac cctttggcaa aatcctgtat atcgtgcgaa aaaggatgga
     2641 tataccgaaa aaatcgctat aatgaccccg aagcagggtt atgcagcgga aaagcgctgc
     2701 ttccctgctg ttttgtggaa tatctaccga ctggaaacag gcaaatgcag gaaattactg
     2761 aactgagggg acaggcgaga gacgatgcca aagagctaca ccgacgagct ggccgagtgg
     2821 gttgaatccc gcgcggccaa gaagcgccgg cgtgatgagg ctgcggttgc gttcctggcg
     2881 gtgagggcgg atgtcgatat gcgtaaggag aaaataccgc atcaggcgca tatttgaatg
     2941 tatttagaaa aataaacaaa aagagtttgt agaaacgcaa aaaggccatc cgtcaggatg
     3001 gccttctgct taatttgatg cctggcagtt tatggcgggc gtcctgcccg ccaccctccg
     3061 ggccgttgct tcgcaacgtt caaatccgct cccggcggat ttgtcctact caggagagcg
     3121 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt
     3181 atttgatgcc tggcagttcc ctactctcgc atggggagac cccacactac catcggcgct
     3241 acggcgtttc acttctgagt tcggcatggg gtcaggtggg accaccgcgc tactgccgcc
     3301 aggcaaattc tgttttatca gaccgcttct gcgttctgat ttaatctgta tcaggctgaa
     3361 aatcttctct catccgccaa aacagccaag ctcgccattc gccattcagg ctgcgcaact
     3421 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     3481 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     3541 cgacggccag tgccaagctt gcatgcctgc aggtcgactc tagaggatcc ccgggtaccg
     3601 agctcgaatt cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3661 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     3721 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3781 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3841 tctccgcata agatgatctt cttgagatcg ttttggtctg cgcgtaatct cttgctctga
     3901 aaacgaaaaa accgccttgc agggcggttt ttcgaaggtt ctctgagcta ccaactcttt
     3961 gaaccgaggt aactggcttg gaggagcgca gtcaccaaaa cttgtccttt cagtttagcc
     4021 ttaaccggcg catgacttca agactaactc ctctaaatca attaccagtg gctgctgcca
     4081 gtggtgcttt tgcatgtctt tccgggttgg actcaagacg atagttaccg gataaggcgc
     4141 agcggtcgga ctgaacgggg ggttcgtgca tacagtccag cttggagcga actgcctacc
     4201 cggaactgag tgtcaggcgt ggaatgagac aaacgcggcc ataacagcgg aatgacaccg
     4261 gtaaaccgaa aggcaggaac aggagagcgc acgagggagc cgccagggga aacgcctggt
     4321 atctttatag tcctgtcggg tttcgccacc actgatttga gcgtcagatt tcgtgatgct
     4381 tgtcaggggg gcggagccta tggaaaaacg gctttgccgc ggccctctca cttccctgtt
     4441 aagtatcttc ctggcatctt ccaggaaatc tccgccccgt tcgtaagcca tttccgctcg
     4501 ccgcagtcga acgaccgagc gtagcgagtc agtgagcgag gaagcggaat atatcctgta
     4561 tcacatattc tgctgacgca ccggtgcagc cttttttctc ctgccacatg aagccctttt
     4621 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacaa
     4681 tcgatgcgaa ttggccgcgg cgttgtgaca atttaccgaa caactccgcg gccgggaagc
     4741 cgatctcggc ttgaacgaat tgttaggtgg cggtacttgg gtcgatatca aagtgcatca
     4801 cttcttcccg tatgcccaac tttgtataga gagccactgc gggatcgtca ccgtaatctg
     4861 cttgcacgta gatcacataa gcaccaagcg cgttggcctc atgcttgagg agattgatga
     4921 gcgcggtggc aatgccctgc ctccggtgct cgccggagac tgcgagatca tagatataga
     4981 tctcactacg cggctgctca aacctgggca gaacgtaagc cgcgagagcg ccaacaaccg
     5041 cttcttggtc gaaggcagca agcgcgatga atgtcttact acggagcaag ttcccgaggt
     5101 aatcggagtc cggctgatgt tgggagtagg tggctacgtc tccgaactca cgaccgaaaa
     5161 gatcaagagc agcccgcatg gatttgactt ggtcagggcc gagcctacat gtgcgaatga
     5221 tgcccatact tgagccacct aactttgttt tagggcgact gccctgctgc gtaacatcgt
     5281 tgctgctgcg taacatcgtt gctgctccat aacatcaaac atcgacccac ggcgtaacgc
     5341 gcttgctgct tggatgcccg aggcatagac tgtacaaaaa aacagtcata acaagccatg
     5401 aaaaccgcca ctgcgccgtt accaccgctg cgttcggtca aggttctgga ccagttgcgt
     5461 gagcgcatac gctacttgca ttacagttta cgaaccgaac aggcttatgt caattcggtt
     5521 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5581 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5641 ttccccgaaa agtgggtaat aactgatata attaaattga agctctaatt tgtgagttta
     5701 gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat cttctcaaat
     5761 atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc cttccctttg
     5821 caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca tcatccacgg
     5881 ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg ggtgtcataa
     5941 tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata aagccgataa
     6001 caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct ccagtagata
     6061 gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc tttgttactt
     6121 cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg tgtgcattcg
     6181 taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat ttgactgtat
     6241 taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg gcggataatg
     6301 cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc acatgtgttt
     6361 ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc ttggtggtac
     6421 gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta aatagcttgg
     6481 cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc gacatgattt
     6541 atcttcgttt cctgcaggtt tttgttctgt gcagttgggt taagaatact gggcaatttc
     6601 atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc tccttccttc
     6661 gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaagaaac cgaaatcaaa
     6721 aaaaagaata aaaaaaaaat gatgaattga attgaaaagc tagcttatcg atgggtcctt
     6781 ttcatcacgt gctataaaaa taattataat ttaaattttt taatataaat atataaatta
     6841 aaaatagaaa gtaaaaaaag aaattaaaga aaaaatagtt tttgttttcc gaagatgtaa
     6901 aagactctag ggggatcgcc aacaaatact accttttatc ttgctcttcc tgctctcagg
     6961 tattaatgcc gaattgtttc atcttgtctg tgtagaagac cacacacgaa aatcctgtga
     7021 ttttacattt tacttatcgt taatcgaatg tatatctatt taatctgctt ttcttgtcta
     7081 ataaatatat atgtaaagta cgctttttgt tgaaattttt taaacctttg tttatttttt
     7141 tttcttcatt ccgtaactct tctaccttct ttatttactt tctaaaatcc aaatacaaaa
     7201 cataaaaata aataaacaca gagtaaattc ccaaattatt ccatcattaa aagatacgag
     7261 gcgcgtgtaa gttacaggca agcgatctct aagaaaccat tattatcatg acattaacct
     7321 ataaaaatag gcgtatcacg aggccctttc gtc
//
