LOCUS       40924_32025        6996 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle vector pMQ175, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6996)
  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 6996)
  AUTHORS   Shanks RMQ., Kadouri DE, O'Toole GA.
  TITLE     Recombineering vectors for a wide range of Gram-negative and 
            -positive bacteria
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 6996)
  AUTHORS   Shanks RMQ., Kadouri DE, O'Toole GA.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-OCT-2008) Ophthalmololgy, University of Pittsburgh, 
            203 Lothrop St, Pittsburgh, PA 15213, USA
REFERENCE   4  (bases 1 to 6996)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 6996)
  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            "Unpublished"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 
            (13-OCT-2008) Ophthalmololgy, University of Pittsburgh, 203 Lothrop 
            St, Pittsburgh, PA 15213, USA"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..801
                     /codon_start=1
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     oriT            complement(893..1001)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     promoter        complement(1192..1210)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      1419..1807
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     terminator      complement(1890..1976)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     primer_bind     2312..2328
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2338..2356
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    complement(2365..2472)
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     promoter        complement(2485..2503)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(2546..2830)
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     CDS             2857..3732
                     /codon_start=1
                     /label=araC
                     /note="L-arabinose regulatory protein"
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     rep_origin      4091..4635
                     /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(5098..5628)
                     /codon_start=1
                     /label=GmR
                     /note="gentamycin acetyltransferase"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(5817..5845)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        complement(5863..5971)
                     /label=AmpR promoter
     misc_feature    6008..6511
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
     promoter        6776..6996
                     /label=URA3 promoter
ORIGIN
        1 atgtcgaaag ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag
       61 ctatttaata tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc
      121 accaaggaat tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca
      181 catgtggata tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta
      241 tccgccaagt acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca
      301 gtcaaattgc agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat
      361 gcacacggtg tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta
      421 acaaaggaac ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct
      481 actggagaat atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc
      541 ggctttattg ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg
      601 acacccggtg tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg
      661 gatgatgtgg tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag
      721 ggaagggatg ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg
      781 agaagatgcg gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac
      841 tcacaaatta gagcttcaat ttaattatat cagttattca cgtggagctt atcggccagc
      901 ctcgcagagc aggattcccg ttgagcaccg ccaggtgcga ataagggaca gtgaagaagg
      961 aacacccgct cgcgggtggg cctacttcac ctatcctgcc cggctgacgc cgttggatac
     1021 accaaggaaa gtctacacga accctttggc aaaatcctgt atatcgtgcg aaaaaggatg
     1081 gatataccga aaaaatcgct ataatgaccc cgaagcaggg ttatgcagcg gaaagtatac
     1141 cttaaccgcc ggctgacccg gcggggacga ggcaagctaa acagatcgac ccctatagtg
     1201 agtcgtatta caaactggaa caacactcac cctatctcgg tctattcttt tgatttataa
     1261 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     1321 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     1381 gcggaaccag atccggccac gatgcgtccg gcgtagagaa tctgaagatc agcagttcaa
     1441 cctgttgata gtacgtacta agctctgatg tttcacgtac taagctctca tgtttaacgt
     1501 actaagctct catgtttaac gaactaaacc ctcatggcta acgtactaag ctctcatggc
     1561 taacgtacta agctctcatg tttcacgtac taagctctca tgtttgaaca ataaaattaa
     1621 tataaatcag caacttaaat agcctccaag gttttaagtt ttataagaaa aaaaagaata
     1681 tataaggctt ttaaagcttt taaggtttaa cggttgtgga caacaagcca gggatgtaac
     1741 gcactgagaa gcccttagag cctctcaaag caattttcag tgacacagga acacttaacg
     1801 gctgacagat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc
     1861 ttcgcaacgt tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac
     1921 aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc
     1981 ctggcagttc cctactctcg catggggaga ccccacacta ccatcggcgc tacggcgttt
     2041 cacttctgag ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc caggcaaatt
     2101 ctgttttatc agaccgcttc tgcgttctga tttaatctgt atcaggctga aaatcttctc
     2161 tcatccgcca aagtttcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     2221 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     2281 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa
     2341 tacgactcac tatagggcga attgggtacc gggccccccc tcgaggtcga cggtatcgat
     2401 aagcttgata tcgaattcct gcagcccggg ggatccacta gttctagagc ggccgccacc
     2461 gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     2521 atggtcattt tttcctccta cgggtatgga gaaacagtag agagttgcga taaaaagcgt
     2581 caggtaggat ccgctaatct tatggataaa aatgctatgg catagcaaag tgtgacgccg
     2641 tgcaaataat caatgtggac ttttctgccg tgattataga cacttttgtt acgcgttttt
     2701 gtcatggctt tggtcccgct ttgttacaga atgcttttaa taagcggggt taccggtttg
     2761 gttagcgaga agagccagta aaagacgcag tgacggcaat gtctgatgca atatggacaa
     2821 ttggtttctt ctctgaatgg cgggagtatg aaaagtatgg ctgaagcgca aaatgatccc
     2881 ctgctgccgg gatactcgtt taatgcccat ctggtggcgg gtttaacgcc gattgaggcc
     2941 aacggttatc tcgatttttt tatcgaccga ccgctgggaa tgaaaggtta tattctcaat
     3001 ctcaccattc gcggtcaggg ggtggtgaaa aatcagggac gagaatttgt ttgccgaccg
     3061 ggtgatattt tgctgttccc gccaggagag attcatcact acggtcgtca tccggaggct
     3121 cgcgaatggt atcaccagtg ggtttacttt cgtccgcgcg cctactggca tgaatggctt
     3181 aactggccgt caatatttgc caatacgggg ttctttcgcc cggatgaagc gcaccagccg
     3241 catttcagcg acctgtttgg gcaaatcatt aacgccgggc aaggggaagg gcgctattcg
     3301 gagctgctgg cgataaatct gcttgagcaa ttgttactgc ggcgcatgga agcgattaac
     3361 gagtcgctcc atccaccgat ggataatcgg gtacgcgagg cttgtcagta catcagcgat
     3421 cacctggcag acagcaattt tgatatcgcc agcgtcgcac agcatgtttg cttgtcgccg
     3481 tcgcgtctgt cacatctttt ccgccagcag ttagggatta gcgtcttaag ctggcgcgag
     3541 gaccaacgta tcagccaggc gaagctgctt ttgagcacca cccggatgcc tatcgccacc
     3601 gtcggtcgca atgttggttt tgacgatcaa ctctatttct cgcgggtatt taaaaaatgc
     3661 accggggcca gcccgagcga gttccgtgcc ggttgtgaag aaaaagtgaa tgatgtagcc
     3721 gtcaagttgt cataattggt aacgaatcag acaattgacg gcttgacgga gtagcatagg
     3781 gtttgcagaa tccctgcttc gtccatttga caggcacatt atgcatcgat taagctgtca
     3841 aacatgagcc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3901 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3961 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4021 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtcagc tgccgcataa
     4081 gatgatcttc ttgagatcgt tttggtctgc gcgtaatctc ttgctctgaa aacgaaaaaa
     4141 ccgccttgca gggcggtttt tcgaaggttc tctgagctac caactctttg aaccgaggta
     4201 actggcttgg aggagcgcag tcaccaaaac ttgtcctttc agtttagcct taaccggcgc
     4261 atgacttcaa gactaactcc tctaaatcaa ttaccagtgg ctgctgccag tggtgctttt
     4321 gcatgtcttt ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcggac
     4381 tgaacggggg gttcgtgcat acagtccagc ttggagcgaa ctgcctaccc ggaactgagt
     4441 gtcaggcgtg gaatgagaca aacgcggcca taacagcgga atgacaccgg taaaccgaaa
     4501 ggcaggaaca ggagagcgca cgagggagcc gccaggggaa acgcctggta tctttatagt
     4561 cctgtcgggt ttcgccacca ctgatttgag cgtcagattt cgtgatgctt gtcagggggg
     4621 cggagcctat ggaaaaacgg ctttgccgcg gccctctcac ttccctgtta agtatcttcc
     4681 tggcatcttc caggaaatct ccgccccgtt cgtaagccat ttccgctcgc cgcagtcgaa
     4741 cgaccgagcg tagcgagtca gtgagcgagg aagcggaata tatcctgtat cacatattct
     4801 gctgacgcac cggtgcagcc ttttttctcc tgccacatga agcaagatcc tttgatcttt
     4861 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     4921 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     4981 taaagtatat atgagtaaac ttggtctgac aatcgatgcg aattggccgc ggcgttgtga
     5041 caatttaccg aacaactccg cggccgggaa gccgatctcg gcttgaacga attgttaggt
     5101 ggcggtactt gggtcgatat caaagtgcat cacttcttcc cgtatgccca actttgtata
     5161 gagagccact gcgggatcgt caccgtaatc tgcttgcacg tagatcacat aagcaccaag
     5221 cgcgttggcc tcatgcttga ggagattgat gagcgcggtg gcaatgccct gcctccggtg
     5281 ctcgccggag actgcgagat catagatata gatctcacta cgcggctgct caaacctggg
     5341 cagaacgtaa gccgcgagag cgccaacaac cgcttcttgg tcgaaggcag caagcgcgat
     5401 gaatgtctta ctacggagca agttcccgag gtaatcggag tccggctgat gttgggagta
     5461 ggtggctacg tctccgaact cacgaccgaa aagatcaaga gcagcccgca tggatttgac
     5521 ttggtcaggg ccgagcctac atgtgcgaat gatgcccata cttgagccac ctaactttgt
     5581 tttagggcga ctgccctgct gcgtaacatc gttgctgctg cgtaacatcg ttgctgctcc
     5641 ataacatcaa acatcgaccc acggcgtaac gcgcttgctg cttggatgcc cgaggcatag
     5701 actgtacaaa aaaacagtca taacaagcca tgaaaaccgc cactgcgccg ttaccaccgc
     5761 tgcgttcggt caaggttctg gaccagttgc gtgagcgcat acgctacttg cattacagtt
     5821 tacgaaccga acaggcttat gtcaattcgg ttgaatactc atactcttcc tttttcaata
     5881 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5941 gaaaaaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgggt
     6001 ccttttcatc acgtgctata aaaataatta taatttaaat tttttaatat aaatatataa
     6061 attaaaaata gaaagtaaaa aaagaaatta aagaaaaaat agtttttgtt ttccgaagat
     6121 gtaaaagact ctagggggat cgccaacaaa tactaccttt tatcttgctc ttcctgctct
     6181 caggtattaa tgccgaattg tttcatcttg tctgtgtaga agaccacaca cgaaaatcct
     6241 gtgattttac attttactta tcgttaatcg aatgtatatc tatttaatct gcttttcttg
     6301 tctaataaat atatatgtaa agtacgcttt ttgttgaaat tttttaaacc tttgtttatt
     6361 tttttttctt cattccgtaa ctcttctacc ttctttattt actttctaaa atccaaatac
     6421 aaaacataaa aataaataaa cacagagtaa attcccaaat tattccatca ttaaaagata
     6481 cgaggcgcgt gtaagttaca ggcaagcgat ccgtcctaag aaaccattat tatcatgaca
     6541 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     6601 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     6661 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     6721 cttaactatg cggcatcaga gcagattgta ctgagagtgc accataccac agcttttcaa
     6781 ttcaattcat catttttttt ttattctttt ttttgatttc ggtttctttg aaattttttt
     6841 gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg agcacagact tagattggta
     6901 tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc cagtattctt aacccaactg
     6961 cacagaacaa aaacctgcag gaaacgaaga taaatc
//
