LOCUS       40924_32020        6032 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pMQ156, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6032)
  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 6032)
  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  JOURNAL   Unpublished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 6032)
  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 6032)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 6032)
  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            "Unpublished"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..6032
                     /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     3115..3131
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    complement(3135..3191)
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3201..3217)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3225..3241)
                     /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(3249..3279)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3294..3315)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        complement(3366..3384)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      3593..3981
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     CDS             complement(4134..4664)
                     /codon_start=1
                     /label=GmR
                     /note="gentamycin acetyltransferase"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(4853..4881)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        complement(4899..5007)
                     /label=AmpR promoter
     misc_feature    5044..5547
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
     promoter        5812..6032
                     /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 aacagccaag ctcgccattc gccattcagg ctgcgcaact
     3001 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     3061 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     3121 cgacggccag tgccaagctt gcatgcctgc aggtcgactc tagaggatcc ccgggtaccg
     3181 agctcgaatt cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3241 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     3301 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaaccgctct
     3361 agacccctat agtgagtcgt attacaaact ggaacaacac tcaccctatc tcggtctatt
     3421 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3481 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     3541 tcggggaaat gtgcgcggaa ccagatccgg ccacgatgcg tccggcgtag agaatctgaa
     3601 gatcagcagt tcaacctgtt gatagtacgt actaagctct gatgtttcac gtactaagct
     3661 ctcatgttta acgtactaag ctctcatgtt taacgaacta aaccctcatg gctaacgtac
     3721 taagctctca tggctaacgt actaagctct catgtttcac gtactaagct ctcatgtttg
     3781 aacaataaaa ttaatataaa tcagcaactt aaatagcctc caaggtttta agttttataa
     3841 gaaaaaaaag aatatataag gcttttaaag cttttaaggt ttaacggttg tggacaacaa
     3901 gccagggatg taacgcactg agaagccctt agagcctctc aaagcaattt tgagtgacac
     3961 aggaacactt aacggctgac agccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4021 gtatatatga gtaaacttgg tctgacaatc gatgcgaatt ggccgcggcg ttgtgacaat
     4081 ttaccgaaca actccgcggc cgggaagccg atctcggctt gaacgaattg ttaggtggcg
     4141 gtacttgggt cgatatcaaa gtgcatcact tcttcccgta tgcccaactt tgtatagaga
     4201 gccactgcgg gatcgtcacc gtaatctgct tgcacgtaga tcacataagc accaagcgcg
     4261 ttggcctcat gcttgaggag attgatgagc gcggtggcaa tgccctgcct ccggtgctcg
     4321 ccggagactg cgagatcata gatatagatc tcactacgcg gctgctcaaa cctgggcaga
     4381 acgtaagccg cgagagcgcc aacaaccgct tcttggtcga aggcagcaag cgcgatgaat
     4441 gtcttactac ggagcaagtt cccgaggtaa tcggagtccg gctgatgttg ggagtaggtg
     4501 gctacgtctc cgaactcacg accgaaaaga tcaagagcag cccgcatgga tttgacttgg
     4561 tcagggccga gcctacatgt gcgaatgatg cccatacttg agccacctaa ctttgtttta
     4621 gggcgactgc cctgctgcgt aacatcgttg ctgctgcgta acatcgttgc tgctccataa
     4681 catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg gatgcccgag gcatagactg
     4741 tacaaaaaaa cagtcataac aagccatgaa aaccgccact gcgccgttac caccgctgcg
     4801 ttcggtcaag gttctggacc agttgcgtga gcgcatacgc tacttgcatt acagtttacg
     4861 aaccgaacag gcttatgtca attcggttga atactcatac tcttcctttt tcaatattat
     4921 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4981 aaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgggtcctt
     5041 ttcatcacgt gctataaaaa taattataat ttaaattttt taatataaat atataaatta
     5101 aaaatagaaa gtaaaaaaag aaattaaaga aaaaatagtt tttgttttcc gaagatgtaa
     5161 aagactctag ggggatcgcc aacaaatact accttttatc ttgctcttcc tgctctcagg
     5221 tattaatgcc gaattgtttc atcttgtctg tgtagaagac cacacacgaa aatcctgtga
     5281 ttttacattt tacttatcgt taatcgaatg tatatctatt taatctgctt ttcttgtcta
     5341 ataaatatat atgtaaagta cgctttttgt tgaaattttt taaacctttg tttatttttt
     5401 tttcttcatt ccgtaactct tctaccttct ttatttactt tctaaaatcc aaatacaaaa
     5461 cataaaaata aataaacaca gagtaaattc ccaaattatt ccatcattaa aagatacgag
     5521 gcgcgtgtaa gttacaggca agcgatccgt cctaagaaac cattattatc atgacattaa
     5581 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     5641 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     5701 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     5761 actatgcggc atcagagcag attgtactga gagtgcacca taccacagct tttcaattca
     5821 attcatcatt ttttttttat tctttttttt gatttcggtt tctttgaaat ttttttgatt
     5881 cggtaatctc cgaacagaag gaagaacgaa ggaaggagca cagacttaga ttggtatata
     5941 tacgcatatg tagtgttgaa gaaacatgaa attgcccagt attcttaacc caactgcaca
     6001 gaacaaaaac ctgcaggaaa cgaagataaa tc
//
