LOCUS       40924_32000        7785 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pMQ125, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7785)
  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 7785)
  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  JOURNAL   Unpublished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 7785)
  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 7785)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 7785)
  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            "Unpublished"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..7785
                     /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"
     protein_bind    complement(1198..1231)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(1282..1633)
                     /direction=LEFT
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     CDS             1647..2477
                     /codon_start=1
                     /label=pRO1600 Rep
                     /note="replication protein for the broad-host-range plasmid
                     pRO1600 from Pseudomonas aeruginosa"
                     /translation="VASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     terminator      complement(2679..2765)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     primer_bind     3101..3117
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3127..3145
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    complement(3154..3261)
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     promoter        complement(3274..3292)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(3335..3619)
                     /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             3646..4521
                     /codon_start=1
                     /label=araC
                     /note="L-arabinose regulatory protein"
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     rep_origin      4880..5424
                     /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(5887..6417)
                     /codon_start=1
                     /label=GmR
                     /note="gentamycin acetyltransferase"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(6606..6634)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        complement(6652..6760)
                     /label=AmpR promoter
     misc_feature    6797..7300
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
     promoter        7565..7785
                     /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 acagatctct agacctaata
     1201 acttcgtata gcatacatta tacgaagtta tattaagggt tgtcgagccg ctggtgccgc
     1261 tggttggacg ccaagggtga atccgcctcg ataccctgat tactcgcttc ctgcgccctc
     1321 tcaggcggcg ataggggact ggtaaaacgg ggattgccca gacgcctccc ccgccccttc
     1381 aggggcacaa atgcggcccc aacggggcca cgtagtggtg cgttttttgc gtttccaccc
     1441 ttttcttcct tttccctttt aaacctttta ggacgtctac aggccacgta atccgtggcc
     1501 tgtagagttt aaaaagggac ggatttgttg ccattaaggg acggatttgt tgttaagaag
     1561 ggacggattt gttgttgtaa agggacggat ttgttgtatt gtgggacgca gatacagtgt
     1621 ccccttatac acaaggaatg tcgaacgtgg cctcaccccc aatggtttac aaaagcaatg
     1681 ccctggtcga ggccgcgtat cgcctcagtg ttcaggaaca gcggatcgtt ctggcctgta
     1741 ttagccaggt gaagaggagc gagcctgtca ccgatgaagt gatgtattca gtgacggcgg
     1801 aggacatagc gacgatggcg ggtgtcccta tcgaatcttc ctacaaccag ctcaaagaag
     1861 cggccctgcg cctgaaacgg cgggaagtcc ggttaaccca agagcccaat ggcaagggga
     1921 aaagaccgag tgtgatgatt accggctggg tgcaaacaat catctaccgg gagggtgagg
     1981 gccgtgtaga actcaggttc accaaagaca tgctgccgta cctgacggaa ctcaccaaac
     2041 agttcaccaa atacgccttg gctgacgtgg ccaagatgga cagcacccac gcgatcaggc
     2101 tttacgagct gctcatgcaa tgggacagca tcggccagcg cgaaatagaa attgaccagc
     2161 tgcgaaagtg gtttcaactg gaaggccggt atccctcgat caaggacttc aagttgcgag
     2221 tgcttgatcc agccgtgacg cagatcaacg agcacagccc gctacaggtg gagtgggcgc
     2281 agcgaaagac cgggcgcaag gtcacacatc tgttgttcag ttttggaccg aagaagcccg
     2341 ccaaggcggt gggtaaggcc ccagcgaagc gcaaggccgg gaagatttca gatgctgaga
     2401 tcgcgaaaca ggctcgccct ggtgagacat gggaagcggc ccgcgctcga ctaacccaga
     2461 tgccgctgga tctggcctag aggccgtggc caccacggcc cggcctgcct ttcaggctgc
     2521 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     2581 ggggatgtgc tgcaaggatg cctggcagtt tatggcgggc gtcctgcccg ccaccctccg
     2641 ggccgttgct tcgcaacgtt caaatccgct cccggcggat ttgtcctact caggagagcg
     2701 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt
     2761 atttgatgcc tggcagttcc ctactctcgc atggggagac cccacactac catcggcgct
     2821 acggcgtttc acttctgagt tcggcatggg gtcaggtggg accaccgcgc tactgccgcc
     2881 aggcaaattc tgttttatca gaccgcttct gcgttctgat ttaatctgta tcaggctgaa
     2941 aatcttctct catccgccaa agtttcgcca ttcaggctgc gcaactgttg ggaagggcga
     3001 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     3061 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag
     3121 cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac
     3181 ggtatcgata agcttgatat cgaattcctg cagcccgggg gatccactag ttctagagcg
     3241 gccgccaccg cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt
     3301 ggcgtaatca tggtcatttt ttcctcctac gggtatggag aaacagtaga gagttgcgat
     3361 aaaaagcgtc aggtaggatc cgctaatctt atggataaaa atgctatggc atagcaaagt
     3421 gtgacgccgt gcaaataatc aatgtggact tttctgccgt gattatagac acttttgtta
     3481 cgcgtttttg tcatggcttt ggtcccgctt tgttacagaa tgcttttaat aagcggggtt
     3541 accggtttgg ttagcgagaa gagccagtaa aagacgcagt gacggcaatg tctgatgcaa
     3601 tatggacaat tggtttcttc tctgaatggc gggagtatga aaagtatggc tgaagcgcaa
     3661 aatgatcccc tgctgccggg atactcgttt aatgcccatc tggtggcggg tttaacgccg
     3721 attgaggcca acggttatct cgattttttt atcgaccgac cgctgggaat gaaaggttat
     3781 attctcaatc tcaccattcg cggtcagggg gtggtgaaaa atcagggacg agaatttgtt
     3841 tgccgaccgg gtgatatttt gctgttcccg ccaggagaga ttcatcacta cggtcgtcat
     3901 ccggaggctc gcgaatggta tcaccagtgg gtttactttc gtccgcgcgc ctactggcat
     3961 gaatggctta actggccgtc aatatttgcc aatacggggt tctttcgccc ggatgaagcg
     4021 caccagccgc atttcagcga cctgtttggg caaatcatta acgccgggca aggggaaggg
     4081 cgctattcgg agctgctggc gataaatctg cttgagcaat tgttactgcg gcgcatggaa
     4141 gcgattaacg agtcgctcca tccaccgatg gataatcggg tacgcgaggc ttgtcagtac
     4201 atcagcgatc acctggcaga cagcaatttt gatatcgcca gcgtcgcaca gcatgtttgc
     4261 ttgtcgccgt cgcgtctgtc acatcttttc cgccagcagt tagggattag cgtcttaagc
     4321 tggcgcgagg accaacgtat cagccaggcg aagctgcttt tgagcaccac ccggatgcct
     4381 atcgccaccg tcggtcgcaa tgttggtttt gacgatcaac tctatttctc gcgggtattt
     4441 aaaaaatgca ccggggccag cccgagcgag ttccgtgccg gttgtgaaga aaaagtgaat
     4501 gatgtagccg tcaagttgtc ataattggta acgaatcaga caattgacgg cttgacggag
     4561 tagcataggg tttgcagaat ccctgcttcg tccatttgac aggcacatta tgcatcgatt
     4621 aagctgtcaa acatgagccc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4681 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4741 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4801 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtcagct
     4861 gccgcataag atgatcttct tgagatcgtt ttggtctgcg cgtaatctct tgctctgaaa
     4921 acgaaaaaac cgccttgcag ggcggttttt cgaaggttct ctgagctacc aactctttga
     4981 accgaggtaa ctggcttgga ggagcgcagt caccaaaact tgtcctttca gtttagcctt
     5041 aaccggcgca tgacttcaag actaactcct ctaaatcaat taccagtggc tgctgccagt
     5101 ggtgcttttg catgtctttc cgggttggac tcaagacgat agttaccgga taaggcgcag
     5161 cggtcggact gaacgggggg ttcgtgcata cagtccagct tggagcgaac tgcctacccg
     5221 gaactgagtg tcaggcgtgg aatgagacaa acgcggccat aacagcggaa tgacaccggt
     5281 aaaccgaaag gcaggaacag gagagcgcac gagggagccg ccaggggaaa cgcctggtat
     5341 ctttatagtc ctgtcgggtt tcgccaccac tgatttgagc gtcagatttc gtgatgcttg
     5401 tcaggggggc ggagcctatg gaaaaacggc tttgccgcgg ccctctcact tccctgttaa
     5461 gtatcttcct ggcatcttcc aggaaatctc cgccccgttc gtaagccatt tccgctcgcc
     5521 gcagtcgaac gaccgagcgt agcgagtcag tgagcgagga agcggaatat atcctgtatc
     5581 acatattctg ctgacgcacc ggtgcagcct tttttctcct gccacatgaa gcaagatcct
     5641 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5701 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5761 aaatcaatct aaagtatata tgagtaaact tggtctgaca atcgatgcga attggccgcg
     5821 gcgttgtgac aatttaccga acaactccgc ggccgggaag ccgatctcgg cttgaacgaa
     5881 ttgttaggtg gcggtacttg ggtcgatatc aaagtgcatc acttcttccc gtatgcccaa
     5941 ctttgtatag agagccactg cgggatcgtc accgtaatct gcttgcacgt agatcacata
     6001 agcaccaagc gcgttggcct catgcttgag gagattgatg agcgcggtgg caatgccctg
     6061 cctccggtgc tcgccggaga ctgcgagatc atagatatag atctcactac gcggctgctc
     6121 aaacctgggc agaacgtaag ccgcgagagc gccaacaacc gcttcttggt cgaaggcagc
     6181 aagcgcgatg aatgtcttac tacggagcaa gttcccgagg taatcggagt ccggctgatg
     6241 ttgggagtag gtggctacgt ctccgaactc acgaccgaaa agatcaagag cagcccgcat
     6301 ggatttgact tggtcagggc cgagcctaca tgtgcgaatg atgcccatac ttgagccacc
     6361 taactttgtt ttagggcgac tgccctgctg cgtaacatcg ttgctgctgc gtaacatcgt
     6421 tgctgctcca taacatcaaa catcgaccca cggcgtaacg cgcttgctgc ttggatgccc
     6481 gaggcataga ctgtacaaaa aaacagtcat aacaagccat gaaaaccgcc actgcgccgt
     6541 taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata cgctacttgc
     6601 attacagttt acgaaccgaa caggcttatg tcaattcggt tgaatactca tactcttcct
     6661 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6721 atgtatttag aaaaaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6781 cacctgggtc cttttcatca cgtgctataa aaataattat aatttaaatt ttttaatata
     6841 aatatataaa ttaaaaatag aaagtaaaaa aagaaattaa agaaaaaata gtttttgttt
     6901 tccgaagatg taaaagactc tagggggatc gccaacaaat actacctttt atcttgctct
     6961 tcctgctctc aggtattaat gccgaattgt ttcatcttgt ctgtgtagaa gaccacacac
     7021 gaaaatcctg tgattttaca ttttacttat cgttaatcga atgtatatct atttaatctg
     7081 cttttcttgt ctaataaata tatatgtaaa gtacgctttt tgttgaaatt ttttaaacct
     7141 ttgtttattt ttttttcttc attccgtaac tcttctacct tctttattta ctttctaaaa
     7201 tccaaataca aaacataaaa ataaataaac acagagtaaa ttcccaaatt attccatcat
     7261 taaaagatac gaggcgcgtg taagttacag gcaagcgatc cgtcctaaga aaccattatt
     7321 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     7381 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     7441 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     7501 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccataccaca
     7561 gcttttcaat tcaattcatc attttttttt tattcttttt tttgatttcg gtttctttga
     7621 aatttttttg attcggtaat ctccgaacag aaggaagaac gaaggaagga gcacagactt
     7681 agattggtat atatacgcat atgtagtgtt gaagaaacat gaaattgccc agtattctta
     7741 acccaactgc acagaacaaa aacctgcagg aaacgaagat aaatc
//
