LOCUS       40924_32065        7454 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle/allelic-replacement vector pMQ30, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7454)
  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 7454)
  AUTHORS   Shanks RMQ., Caiazza NC, Hinsa SM, Toutain CM, O'Toole GA.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-SEP-2005) Microbiology and Immunology, Dartmouth 
            Medical School, Vail Building Room 505, Hanover, NH 03755, USA
REFERENCE   3  (bases 1 to 7454)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7454)
  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 
            (14-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..7454
                     /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      complement(4023..4611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4867..5397)
                     /codon_start=1
                     /label=GmR
                     /note="gentamycin acetyltransferase"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(5586..5614)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        complement(5632..5736)
                     /label=AmpR promoter
     CDS             complement(5836..6636)
                     /codon_start=1
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(6637..6857)
                     /label=URA3 promoter
     misc_feature    6885..7388
                     /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 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3901 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3961 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4021 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4081 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4141 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4201 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4261 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4321 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4381 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4441 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     4501 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4561 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4621 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4681 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4741 aaatcaatct aaagtatata tgagtaaact tggtctgaca atcgatgcga attggccgcg
     4801 gcgttgtgac aatttaccga acaactccgc ggccgggaag ccgatctcgg cttgaacgaa
     4861 ttgttaggtg gcggtacttg ggtcgatatc aaagtgcatc acttcttccc gtatgcccaa
     4921 ctttgtatag agagccactg cgggatcgtc accgtaatct gcttgcacgt agatcacata
     4981 agcaccaagc gcgttggcct catgcttgag gagattgatg agcgcggtgg caatgccctg
     5041 cctccggtgc tcgccggaga ctgcgagatc atagatatag atctcactac gcggctgctc
     5101 aaacctgggc agaacgtaag ccgcgagagc gccaacaacc gcttcttggt cgaaggcagc
     5161 aagcgcgatg aatgtcttac tacggagcaa gttcccgagg taatcggagt ccggctgatg
     5221 ttgggagtag gtggctacgt ctccgaactc acgaccgaaa agatcaagag cagcccgcat
     5281 ggatttgact tggtcagggc cgagcctaca tgtgcgaatg atgcccatac ttgagccacc
     5341 taactttgtt ttagggcgac tgccctgctg cgtaacatcg ttgctgctgc gtaacatcgt
     5401 tgctgctcca taacatcaaa catcgaccca cggcgtaacg cgcttgctgc ttggatgccc
     5461 gaggcataga ctgtacaaaa aaacagtcat aacaagccat gaaaaccgcc actgcgccgt
     5521 taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata cgctacttgc
     5581 attacagttt acgaaccgaa caggcttatg tcaattcggt tgaatactca tactcttcct
     5641 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5701 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgggtaa
     5761 taactgatat aattaaattg aagctctaat ttgtgagttt agtatacatg catttactta
     5821 taatacagtt ttttagtttt gctggccgca tcttctcaaa tatgcttccc agcctgcttt
     5881 tctgtaacgt tcaccctcta ccttagcatc ccttcccttt gcaaatagtc ctcttccaac
     5941 aataataatg tcagatcctg tagagaccac atcatccacg gttctatact gttgacccaa
     6001 tgcgtctccc ttgtcatcta aacccacacc gggtgtcata atcaaccaat cgtaaccttc
     6061 atctcttcca cccatgtctc tttgagcaat aaagccgata acaaaatctt tgtcgctctt
     6121 cgcaatgtca acagtaccct tagtatattc tccagtagat agggagccct tgcatgacaa
     6181 ttctgctaac atcaaaaggc ctctaggttc ctttgttact tcttctgccg cctgcttcaa
     6241 accgctaaca atacctgggc ccaccacacc gtgtgcattc gtaatgtctg cccattctgc
     6301 tattctgtat acacccgcag agtactgcaa tttgactgta ttaccaatgt cagcaaattt
     6361 tctgtcttcg aagagtaaaa aattgtactt ggcggataat gcctttagcg gcttaactgt
     6421 gccctccatg gaaaaatcag tcaagatatc cacatgtgtt tttagtaaac aaattttggg
     6481 acctaatgct tcaactaact ccagtaattc cttggtggta cgaacatcca atgaagcaca
     6541 caagtttgtt tgcttttcgt gcatgatatt aaatagcttg gcagcaacag gactaggatg
     6601 agtagcagca cgttccttat atgtagcttt cgacatgatt tatcttcgtt tcctgcaggt
     6661 ttttgttctg tgcagttggg ttaagaatac tgggcaattt catgtttctt caacactaca
     6721 tatgcgtata tataccaatc taagtctgtg ctccttcctt cgttcttcct tctgttcgga
     6781 gattaccgaa tcaaaaaaat ttcaaagaaa ccgaaatcaa aaaaaagaat aaaaaaaaaa
     6841 tgatgaattg aattgaaaag ctagcttatc gatgggtcct tttcatcacg tgctataaaa
     6901 ataattataa tttaaatttt ttaatataaa tatataaatt aaaaatagaa agtaaaaaaa
     6961 gaaattaaag aaaaaatagt ttttgttttc cgaagatgta aaagactcta gggggatcgc
     7021 caacaaatac taccttttat cttgctcttc ctgctctcag gtattaatgc cgaattgttt
     7081 catcttgtct gtgtagaaga ccacacacga aaatcctgtg attttacatt ttacttatcg
     7141 ttaatcgaat gtatatctat ttaatctgct tttcttgtct aataaatata tatgtaaagt
     7201 acgctttttg ttgaaatttt ttaaaccttt gtttattttt ttttcttcat tccgtaactc
     7261 ttctaccttc tttatttact ttctaaaatc caaatacaaa acataaaaat aaataaacac
     7321 agagtaaatt cccaaattat tccatcatta aaagatacga ggcgcgtgta agttacaggc
     7381 aagcgatctc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     7441 gaggcccttt cgtc
//
