LOCUS       Exported                5997 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Allelic replacement vector pMQ236, complete sequence.
ACCESSION   GQ160907
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5997)
  AUTHORS   Shanks RM, Kadouri DE, MacEachran DP, OToole GA.
  TITLE     New yeast recombineering tools for bacteria
  JOURNAL   Plasmid 62 (2), 88-97 (2009)
  PUBMED    19477196
REFERENCE   2  (bases 1 to 5997)
  AUTHORS   Shanks RMQ., Kadouri DE, MacEachran DP, OToole GA.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-MAY-2009) Ophthalmology, University of Pittsburgh, 203
            Lothrop St, Pittsburgh, PA 15213, USA
REFERENCE   3  (bases 1 to 5997)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5997
                     /organism="Allelic replacement vector pMQ236"
                     /mol_type="other DNA"
                     /db_xref="taxon:651991"
     misc_feature    6..830
                     /label=oriT
                     /note="oriT"
     oriT            352..461
                     /note="incP origin of transfer"
     terminator      882..909
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      1001..1087
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1243..1527)
                     /codon_start=1
                     /gene="lacZ-alpha"
                     /product="B-galactosidase alpha peptide"
                     /label=lacZ-alpha
                     /protein_id="ACS35605.1"
                     /translation="MTMITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRAWLFWRMREDFQPDTD"
     gene            complement(1243..1527)
                     /gene="lacZ-alpha"
                     /label=lacZ-alpha
     primer_bind     1437..1453
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1457..1513
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1523..1539)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1547..1563
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     regulatory      complement(1566..1649)
                     /regulatory_class="promoter"
                     /note="Plac; derived from Escherichia coli"
     promoter        complement(1571..1601)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1616..1637
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        complement(1688..1706)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      1914..2302
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     rep_origin      1974..2194
                     /label=oriR6K
                     /note="oriR6K"
     CDS             2653..3447
                     /codon_start=1
                     /gene="nptII"
                     /product="KanR marker"
                     /label=nptII
                     /protein_id="ACS35606.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     gene            2653..3447
                     /gene="nptII"
                     /label=nptII
     misc_feature    3463..3492
                     /label=I-SceI meganuclease site
                     /note="I-SceI meganuclease site"
     gene            3701..4135
                     /gene="rpsL"
                     /label=rpsL
     CDS             3761..4135
                     /codon_start=1
                     /gene="rpsL"
                     /product="RpsL"
                     /label=rpsL
                     /note="ribosomal protein confers streptomycin sensitivity"
                     /protein_id="ACS35607.1"
                     /translation="MATVNQLVRKPRARKVAKSNVPALEACPQKRGVCTRVYTTTPKKP
                     NSALRKVCRVRLTNGFEVTSYIGGEGHNLQEHSVILIRGGRVKDLPGVRYHTVRGALDC
                     SGVKDRKQARSKYGVKRPKA"
     CDS             complement(4395..5198)
                     /codon_start=1
                     /gene="ura3"
                     /product="Ura3"
                     /label=ura3
                     /protein_id="ACS35608.1"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     gene            complement(4395..5198)
                     /gene="ura3"
                     /label=ura3
     promoter        complement(5199..5414)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     rep_origin      5437..5950
                     /note="CEN6/ARSH4; derived from Saccharomyces cerevisiae"
     misc_feature    5447..5950
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"