LOCUS       40924_32010        6071 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pMQ132, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6071)
  AUTHORS   Shanks RM, Kadouri DE, MacEachran DP, O'Toole GA.
  TITLE     New yeast recombineering tools for bacteria
  JOURNAL   Plasmid 62 (2), 88-97 (2009)
  PUBMED    19477196
REFERENCE   2  (bases 1 to 6071)
  AUTHORS   Shanks RM, MacEachran DP, Powers SZ, Kadouri DE, O'Toole GA.
  TITLE     In vivo recombination vectors for modification and expression of 
            genes in Gram-negative and Gram-positive bacteria
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 6071)
  AUTHORS   Shanks RM, MacEachran DP, Powers SZ, Kadouri DE, O'Toole GA.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-MAR-2008) Ophthalmology, University of Pittsburgh, 203
            Lothrop St., Pittsburgh, PA 15213, USA
REFERENCE   4  (bases 1 to 6071)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 6071)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plasmid"; 
            date: "2009"; volume: "62"; issue: "2"; pages: "88-97"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted 
            (04-MAR-2008) Ophthalmology, University of Pittsburgh, 203 Lothrop 
            St., Pittsburgh, PA 15213, USA"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            complement(399..508)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     terminator      complement(929..956)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(1048..1134)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     primer_bind     1484..1500
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    complement(1504..1560)
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1570..1586)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(1594..1610)
                     /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(1618..1648)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(1663..1684)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      1834..2603
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             2604..3263
                     /codon_start=1
                     /label=pBBR1 Rep
                     /note="replication protein for the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica"
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     CDS             complement(3550..4080)
                     /codon_start=1
                     /label=GmR
                     /note="gentamycin acetyltransferase"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(4269..4297)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        complement(4315..4419)
                     /label=AmpR promoter
     CDS             complement(4519..5319)
                     /codon_start=1
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(5320..5540)
                     /label=URA3 promoter
     misc_feature    5568..6071
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
ORIGIN
        1 tctaagaaac cattattatc atgacattaa cctataaaaa aggcctctcg agctagagtc
       61 gatcttcgcc agcagggcga ggatcgtggc atcaccgaac cgcgccgtgc gcgggtcgtc
      121 ggtgagccag agtttcagca ggccgcccag gcggcccagg tcgccattga tgcgggccag
      181 ctcgcggacg tgctcatagt ccacgacgcc cgtgattttg tagccctggc cgacggccag
      241 caggtaggcc gacaggctca tgccggccgc cgccgccttt tcctcaatcg ctcttcgttc
      301 gtctggaagg cagtacacct tgataggtgg gctgcccttc ctggttggct tggtttcatc
      361 agccatccgc ttgccctcat ctgttacgcc ggcggtagcc ggccagcctc gcagagcagg
      421 attcccgttg agcaccgcca ggtgcgaata agggacagtg aagaaggaac acccgctcgc
      481 gggtgggcct acttcaccta tcctgcccgg ctgacgccgt tggatacacc aaggaaagtc
      541 tacacgaacc ctttggcaaa atcctgtata tcgtgcgaaa aaggatggat ataccgaaaa
      601 aatcgctata atgaccccga agcagggtta tgcagcggaa aagcgctgct tccctgctgt
      661 tttgtggaat atctaccgac tggaaacagg caaatgcagg aaattactga actgagggga
      721 caggcgagag acgatgccaa agagctacac cgacgagctg gccgagtggg ttgaatcccg
      781 cgcggccaag aagcgccggc gtgatgaggc tgcggttgcg ttcctggcgg tgagggcgga
      841 tgtcgatatg cgtaaggaga aaataccgca tcaggcgcat atttgaatgt atttagaaaa
      901 ataaacaaaa agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt
      961 aatttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt
     1021 cgcaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa
     1081 acaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta tttgatgcct
     1141 ggcagttccc tactctcgca tggggagacc ccacactacc atcggcgcta cggcgtttca
     1201 cttctgagtt cggcatgggg tcaggtggga ccaccgcgct actgccgcca ggcaaattct
     1261 gttttatcag accgcttctg cgttctgatt taatctgtat caggctgaaa atcttctctc
     1321 atccgccaaa acagccaagc tcgccattcg ccattcaggc tgcgcaactg ttgggaaggg
     1381 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     1441 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     1501 gccaagcttg catgcctgca ggtcgactct agaggatccc cgggtaccga gctcgaattc
     1561 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     1621 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     1681 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     1741 ttaatgcgcc ctacgggctt gctctccggg cttcgccctg cgcggtcgct gcgctccctt
     1801 gccagcccgt ggatatgtgg acgatggccg cgagcggcca ccggctggct cgcttcgctc
     1861 ggcccgtgga caaccctgct ggacaagctg atggacaggc tgcgcctgcc cacgagcttg
     1921 accacaggga ttgcccaccg gctacccagc cttcgaccac atacccaccg gctccgactg
     1981 cgcggcctgc ggccttgccc catcaatttt tttaattttc tctggggaaa agcctccggc
     2041 ctgcggcctg cgcgcttcgc ttgccggttg gacaccaagt ggaaggcggg tcaaggctcg
     2101 cgcagcgacc gcgcagcggc ttggccttga cgcgcctgga acgacccaag cctatgcgag
     2161 tgggggcagt cgaaggcgaa gcccgcccgc ctgccccccg agcctcacgg cggcgagtgc
     2221 gggggttcca agggggcagc gccaccttgg gcaaggccga aggccgcgca gtcgatcaac
     2281 aagccccgga ggggccactt tttgccggag ggggagccgc gccgaaggcg tgggggaacc
     2341 ccgcaggggt gcccttcttt gggcaccaaa gaactagata tagggcgaaa tgcgaaagac
     2401 ttaaaaatca acaacttaaa aaaggggggt acgcaacagc tcattgcggc accccccgca
     2461 atagctcatt gcgtaggtta aagaaaatct gtaattgact gccactttta cgcaacgcat
     2521 aattgttgtc gcgctgccga aaagttgcag ctgattgcgc atggtgccgc aaccgtgcgg
     2581 caccctaccg catggagata agcatggcca cgcagtccag agaaatcggc attcaagcca
     2641 agaacaagcc cggtcactgg gtgcaaacgg aacgcaaagc gcatgaggcg tgggccgggc
     2701 ttattgcgag gaaacccacg gcggcaatgc tgctgcatca cctcgtggcg cagatgggcc
     2761 accagaacgc cgtggtggtc agccagaaga cactttccaa gctcatcgga cgttctttgc
     2821 ggacggtcca atacgcagtc aaggacttgg tggccgagcg ctggatctcc gtcgtgaagc
     2881 tcaacggccc cggcaccgtg tcggcctacg tggtcaatga ccgcgtggcg tggggccagc
     2941 cccgcgacca gttgcgcctg tcggtgttca gtgccgccgt ggtggttgat cacgacgacc
     3001 aggacgaatc gctgttgggg catggcgacc tgcgccgcat cccgaccctg tatccgggcg
     3061 agcagcaact accgaccggc cccggcgagg agccgcccag ccagcccggc attccgggca
     3121 tggaaccaga cctgccagcc ttgaccgaaa cggaggaatg ggaacggcgc gggcagcagc
     3181 gcctgccgat gcccgatgag ccgtgttttc tggacgatgg cgagccgttg gagccgccga
     3241 cacgggtcac gctgccgcgc cggtagcact tgggttgcgc agcaacccgt aagtgcgctg
     3301 ttccagacta tcggctgtag ccgcctcgcc gccctatacc ttgtctgcct ccccgcgttg
     3361 cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagcctttta cgagtttttg
     3421 gtgcaatcaa tctaaagtat atatgagtaa acttggtctg acaatcgatg cgaattggcc
     3481 gcggcgttgt gacaatttac cgaacaactc cgcggccggg aagccgatct cggcttgaac
     3541 gaattgttag gtggcggtac ttgggtcgat atcaaagtgc atcacttctt cccgtatgcc
     3601 caactttgta tagagagcca ctgcgggatc gtcaccgtaa tctgcttgca cgtagatcac
     3661 ataagcacca agcgcgttgg cctcatgctt gaggagattg atgagcgcgg tggcaatgcc
     3721 ctgcctccgg tgctcgccgg agactgcgag atcatagata tagatctcac tacgcggctg
     3781 ctcaaacctg ggcagaacgt aagccgcgag agcgccaaca accgcttctt ggtcgaaggc
     3841 agcaagcgcg atgaatgtct tactacggag caagttcccg aggtaatcgg agtccggctg
     3901 atgttgggag taggtggcta cgtctccgaa ctcacgaccg aaaagatcaa gagcagcccg
     3961 catggatttg acttggtcag ggccgagcct acatgtgcga atgatgccca tacttgagcc
     4021 acctaacttt gttttagggc gactgccctg ctgcgtaaca tcgttgctgc tgcgtaacat
     4081 cgttgctgct ccataacatc aaacatcgac ccacggcgta acgcgcttgc tgcttggatg
     4141 cccgaggcat agactgtaca aaaaaacagt cataacaagc catgaaaacc gccactgcgc
     4201 cgttaccacc gctgcgttcg gtcaaggttc tggaccagtt gcgtgagcgc atacgctact
     4261 tgcattacag tttacgaacc gaacaggctt atgtcaattc ggttgaatac tcatactctt
     4321 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4381 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtggg
     4441 taataactga tataattaaa ttgaagctct aatttgtgag tttagtatac atgcatttac
     4501 ttataataca gttttttagt tttgctggcc gcatcttctc aaatatgctt cccagcctgc
     4561 ttttctgtaa cgttcaccct ctaccttagc atcccttccc tttgcaaata gtcctcttcc
     4621 aacaataata atgtcagatc ctgtagagac cacatcatcc acggttctat actgttgacc
     4681 caatgcgtct cccttgtcat ctaaacccac accgggtgtc ataatcaacc aatcgtaacc
     4741 ttcatctctt ccacccatgt ctctttgagc aataaagccg ataacaaaat ctttgtcgct
     4801 cttcgcaatg tcaacagtac ccttagtata ttctccagta gatagggagc ccttgcatga
     4861 caattctgct aacatcaaaa ggcctctagg ttcctttgtt acttcttctg ccgcctgctt
     4921 caaaccgcta acaatacctg ggcccaccac accgtgtgca ttcgtaatgt ctgcccattc
     4981 tgctattctg tatacacccg cagagtactg caatttgact gtattaccaa tgtcagcaaa
     5041 ttttctgtct tcgaagagta aaaaattgta cttggcggat aatgccttta gcggcttaac
     5101 tgtgccctcc atggaaaaat cagtcaagat atccacatgt gtttttagta aacaaatttt
     5161 gggacctaat gcttcaacta actccagtaa ttccttggtg gtacgaacat ccaatgaagc
     5221 acacaagttt gtttgctttt cgtgcatgat attaaatagc ttggcagcaa caggactagg
     5281 atgagtagca gcacgttcct tatatgtagc tttcgacatg atttatcttc gtttcctgca
     5341 ggtttttgtt ctgtgcagtt gggttaagaa tactgggcaa tttcatgttt cttcaacact
     5401 acatatgcgt atatatacca atctaagtct gtgctccttc cttcgttctt ccttctgttc
     5461 ggagattacc gaatcaaaaa aatttcaaag aaaccgaaat caaaaaaaag aataaaaaaa
     5521 aaatgatgaa ttgaattgaa aagctagctt atcgatgggt ccttttcatc acgtgctata
     5581 aaaataatta taatttaaat tttttaatat aaatatataa attaaaaata gaaagtaaaa
     5641 aaagaaatta aagaaaaaat agtttttgtt ttccgaagat gtaaaagact ctagggggat
     5701 cgccaacaaa tactaccttt tatcttgctc ttcctgctct caggtattaa tgccgaattg
     5761 tttcatcttg tctgtgtaga agaccacaca cgaaaatcct gtgattttac attttactta
     5821 tcgttaatcg aatgtatatc tatttaatct gcttttcttg tctaataaat atatatgtaa
     5881 agtacgcttt ttgttgaaat tttttaaacc tttgtttatt tttttttctt cattccgtaa
     5941 ctcttctacc ttctttattt actttctaaa atccaaatac aaaacataaa aataaataaa
     6001 cacagagtaa attcccaaat tattccatca ttaaaagata cgaggcgcgt gtaagttaca
     6061 ggcaagcgat c
//
