LOCUS       Exported                9816 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle vector pME6032, complete sequence.
ACCESSION   DQ645594
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9816)
  AUTHORS   Heeb S, Itoh Y, Nishijyo T, Schnider U, Keel C, Wade J, Walsh U, 
            OGara F, Haas D.
  TITLE     Small, stable shuttle vectors based on the minimal pVS1 replicon for
            use in gram-negative, plant-associated bacteria
  JOURNAL   Mol. Plant Microbe Interact. 13 (2), 232-237 (2000)
  PUBMED    10659714
REFERENCE   2  (bases 1 to 9816)
  AUTHORS   Heeb S, Blumer C, Haas D.
  TITLE     Regulatory RNA as mediator in GacA/RsmA-dependent global control of 
            exoproduct formation in Pseudomonas fluorescens CHA0
  JOURNAL   J. Bacteriol. 184 (4), 1046-1056 (2002)
  PUBMED    11807065
REFERENCE   3  (bases 1 to 9816)
  AUTHORS   Heeb S.
  TITLE     Direct Submission
  JOURNAL   Submitted (19-MAY-2006) Institute of Infection, Immunity and 
            Inflammation, University of Nottingham, Centre for Biomolecular 
            Sciences, Nottingham NG7 2RD, United Kingdom
REFERENCE   4  (bases 1 to 9816)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9816
                     /organism="Shuttle vector pME6032"
                     /mol_type="other DNA"
                     /db_xref="taxon:390901"
     CDS             195..881
                     /codon_start=1
                     /product="pVS1 resolvase"
                     /label=pVS1 resolvase
                     /note="ORF1; not required for stable maintenance"
                     /protein_id="ABG33931.1"
                     /translation="MNKSAAAGLLGYARVSTDDQDLTNQRAELHAAGCTKLFSEKITGT
                     RRDRPELARMLDHLRPGDVVTVTRLDRLARSTRDLLDIAERIQEAGAGLRSLAEPWADT
                     TTPAGRMVLTVFAGIAEFERSLIIDRTRSGREAAKARGVKFGPRPTLTPAQIAHARELI
                     DQEGRTVKEAAALLGVHRSTLYRALERSEEVTPTEARRRGAFREDALTEADALAAAENE
                     RQEEQA"
     CDS             878..1093
                     /codon_start=1
                     /product="hypothetical protein"
                     /label=hypothetical protein
                     /note="ORF2; not required for stable maintenance"
                     /protein_id="ABG33932.1"
                     /translation="MKPHQDGQDEPFFITEEIEAEMIAAGYVFEPPAHVSTVRLHEILA
                     GLSDAKLAAWPASLAAEETERRRLKR"
     CDS             1180..1809
                     /codon_start=1
                     /product="StaA"
                     /label=StaA
                     /note="pVS1 partitioning protein"
                     /protein_id="ABG33933.1"
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             1830..2045
                     /codon_start=1
                     /product="ParG"
                     /label=ParG
                     /note="pVS1 partitioning protein"
                     /protein_id="ABG33934.1"
                     /translation="MSKSTNTLSAGRPSARSSKAATLASLADTPAMKRVNFQLPAEDHT
                     KLKMYAVRQGKTITELLSEYIAQLPE"
     CDS             2238..3311
                     /codon_start=1
                     /product="RepA"
                     /label=RepA
                     /note="pVS1 replication protein"
                     /protein_id="ABG33935.1"
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      3377..3571
                     /label=pVS1 origin of replication
                     /note="pVS1 origin of replication"
     rep_origin      4264..5089
                     /label=p15A origin of replication
                     /note="p15A origin of replication"
     rep_origin      4281..4826
                     /direction=RIGHT
                     /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."
     oriT            5068..5089
                     /label=p15A origin of transfer
                     /note="p15A origin of transfer"
     regulatory      5148..5414
                     /regulatory_class="terminator"
                     /note="T4 transcription terminator"
     misc_feature    5402..5421
                     /note="sequencing oligonucleotide P6032: 
                     5-CCCTCACTGATCCGCTAGTC-3"
     misc_feature    5416..5473
                     /label=multiple cloning site
                     /note="multiple cloning site"
     misc_feature    complement(5468..5542)
                     /label=tac promoter, 5UTR and RBS followed by EcoRI
                     /note="tac promoter, 5UTR and RBS followed by EcoRI"
     regulatory      complement(5478..5481)
                     /regulatory_class="ribosome_binding_site"
     protein_bind    5490..5506
                     /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)."
     misc_feature    complement(5508..5527)
                     /note="sequencing oligonucleotide PTAC: 
                     5-CGGCTCGTATAATGTGTGGA-3"
     misc_feature    complement(5509)
                     /label=start of transcription
                     /note="start of transcription"
     promoter        complement(5514..5542)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     regulatory      complement(5514..5520)
                     /regulatory_class="minus_10_signal"
     regulatory      complement(5537..5542)
                     /regulatory_class="minus_35_signal"
     promoter        5776..5853
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             5977..6792
                     /codon_start=1
                     /product="LacIQ repressor"
                     /label=LacIQ repressor
                     /note="tac promoter repressor"
                     /protein_id="ABG33936.1"
                     /translation="MAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSR
                     ADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPA
                     LFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKY
                     LTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLR
                     VGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTTWTACCNSLRARR"
     CDS             complement(7821..8498)
                     /codon_start=1
                     /product="TetR"
                     /label=TetR
                     /note="RK2 tetracycline resistance repressor protein"
                     /protein_id="ABG33937.1"
                     /translation="MFISDKVSSMTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGV
                     QQPALYWHFRNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRD
                     GARIHAGTRPGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSD
                     AGERGGTVEQAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRK
                     GDD"
     CDS             complement(7821..8471)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline resistance regulatory protein"
                     /label=TetR
                     /translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
                     RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
                     PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
                     QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD"
     CDS             8577..9776
                     /codon_start=1
                     /product="TetA"
                     /label=TetA
                     /note="RK2 tetracycline resistance protein"
                     /protein_id="ABG33938.1"
                     /translation="MKPNIPLIVILSTVALDAVGIGLIMPVLPGLLRDLVHSNDVTAHY
                     GILLALYALVQFACAPVLGALSDRFGRRPILLVSLAGATVDYAIMATAPFLWVLYIGRI
                     VAGITGATGAVAGAYIADITDGDERARHFGFMSACFGFGMVAGPVLGGLMGGFSPHAPF
                     FAAAALNGLNFLTGCFLLPESHKGERRPLRREALNPLASFRWARGMTVVAALMAVFFIM
                     QLVGQVPAALWVIFGEDRFHWDATTIGISLAAFGILHSLAQAMITGPVAARLGERRALM
                     LGMIADGTGYILLAFATRGWMAFPIMVLLASGGIGMPALQAMLSRQVDEERQGQLQGSL
                     AALTSLTSIVGPLLFTAIYAASITTWNGWAWIAGAALYLLCLPALRRGLWSGAGQRADR
                     "