LOCUS       Exported               12721 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Binary vector pN6-Bxb1, complete sequence.
ACCESSION   JQ305140
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12721)
  AUTHORS   Thomson JG, Thilmony R.
  TITLE     Direct Submission
  JOURNAL   Submitted (19-DEC-2011) Crop Improvement and Utilization Research 
            Unit, USDA-ARS-WRRC, 800 Buchanan Street, Albany, CA 94710-1105, USA
REFERENCE   2  (bases 1 to 12721)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..12721
                     /organism="Binary vector pN6-Bxb1"
                     /mol_type="other DNA"
                     /db_xref="taxon:1167201"
     misc_feature    complement(1..26)
                     /label=Agrobacterium left border
                     /note="Agrobacterium left border"
     misc_feature    1..25
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     regulatory      complement(76..291)
                     /regulatory_class="polyA_signal_sequence"
                     /note="CaMV 35S polyA terminator"
     polyA_signal    103..277
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(317..1342)
                     /codon_start=1
                     /gene="hptII"
                     /product="hygromycin phosphotransferase II"
                     /label=hptII
                     /note="hygromycin resistance plant selectable marker"
                     /protein_id="AFF61393.1"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     gene            complement(317..1342)
                     /gene="hptII"
                     /label=hptII
     regulatory      complement(1378..1820)
                     /regulatory_class="promoter"
                     /note="double enhanced CaMV 35S promoter"
     promoter        complement(1410..2087)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     protein_bind    2278..2299
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2314..2344
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2352..2368
                     /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)."
     primer_bind     2376..2392
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2388..2621
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEC"
     misc_feature    2402..2458
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2462..2478)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2855..3200
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     3095..3125
                     /label=35S forward e primer annealing site
                     /note="35S forward e primer annealing site"
     misc_recomb     3233..3284
                     /label=Bxb1 recombinase attP attachment site
                     /note="Bxb1 recombinase attP attachment site"
     misc_feature    3285..4039
                     /label=stuffer DNA
                     /note="stuffer DNA"
     CDS             3295..4014
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_recomb     4040..4081
                     /label=Bxb1 recombinase attB attachment site
                     /note="Bxb1 recombinase attB attachment site"
     CDS             join(4089..4103,4294..6141)
                     /codon_start=1
                     /gene="gusA"
                     /product="beta-glucuronidase"
                     /label=gusA
                     /note="GUS reporter"
                     /protein_id="AFF61394.1"
                     /translation="MVDLRNRRLVRPVETPTREIKKLDGLWAFSLDRENCGIDQRWWES
                     ALQESRAIAVPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDAVTHYGK
                     VWVNNQEVMEHQGGYTPFEADVTPYVIAGKSVRITVCVNNELNWQTIPPGMVITDENGK
                     KKQSYFHDFFNYAGIHRSVMLYTTPNTWVDDITVVTHVAQDCNHASVDWQVVANGDVSV
                     ELRDADQQVVATGQGTSGTLQVVNPHLWQPGEGYLYELEVTAKSQTESDIYPLRVGIRS
                     VAVKGQQFLINHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSHYP
                     YAEEMLDWADEHGIVVIDETAAVGFQLSLGIGFEAGNKPKELYSEEAVNGETQQAHLQA
                     IKELIARDKNHPSVVMWSIANEPDTRPQGAREYFAPLAEATRKLDPTRPITCVNVMFCD
                     AHTDTISDLFDVLCLNRYYGWYVQSGDLETAEKVLEKELLAWQEKLHQPIIITEYGVDT
                     LAGLHSMYTDMWSEEYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKK
                     GIFTRDRKPKSAAFLLQKRWTGMNFGEKPQQGGKQASHHHHHHV"
     gene            4089..6141
                     /gene="gusA"
                     /label=gusA
     CDS             join(4089..4103,4294..6111)
                     /codon_start=1
                     /product="beta-glucuronidase"
                     /label=GUS
                     /note="This version of the gusA gene has a 5 extension 
                     with a catalase intron to ensure expression in plants but 
                     not bacteria."
                     /translation="MVDLRNRRLVRPVETPTREIKKLDGLWAFSLDRENCGIDQRWWES
                     ALQESRAIAVPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDAVTHYGK
                     VWVNNQEVMEHQGGYTPFEADVTPYVIAGKSVRITVCVNNELNWQTIPPGMVITDENGK
                     KKQSYFHDFFNYAGIHRSVMLYTTPNTWVDDITVVTHVAQDCNHASVDWQVVANGDVSV
                     ELRDADQQVVATGQGTSGTLQVVNPHLWQPGEGYLYELEVTAKSQTESDIYPLRVGIRS
                     VAVKGQQFLINHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSHYP
                     YAEEMLDWADEHGIVVIDETAAVGFQLSLGIGFEAGNKPKELYSEEAVNGETQQAHLQA
                     IKELIARDKNHPSVVMWSIANEPDTRPQGAREYFAPLAEATRKLDPTRPITCVNVMFCD
                     AHTDTISDLFDVLCLNRYYGWYVQSGDLETAEKVLEKELLAWQEKLHQPIIITEYGVDT
                     LAGLHSMYTDMWSEEYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKK
                     GIFTRDRKPKSAAFLLQKRWTGMNFGEKPQQGGKQ"
     intron          4104..4293
                     /gene="gusA"
                     /note="catalase intron"
     primer_bind     complement(4297..4322)
                     /label=GUS reverse f primer annealing site
                     /note="GUS reverse f primer annealing site"
     CDS             6118..6135
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      6170..6422
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     regulatory      6195..6425
                     /regulatory_class="polyA_signal_sequence"
                     /note="nopaline synthase (nos) polyA terminator"
     misc_feature    6444..6468
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     misc_feature    complement(6463..6488)
                     /label=Agrobacterium right border
                     /note="Agrobacterium right border"
     misc_feature    6489..12721
                     /label=pCAMBIA 1301 binary vector sequence
                     /note="pCAMBIA 1301 binary vector sequence"
     CDS             7768..8397
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             8831..9898
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
                     AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
                     DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
                     VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
                     ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
                     RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
                     MRYRNLIEGEASAGS"
     rep_origin      9964..10158
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     misc_feature    10502..10642
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(10828..11416)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(11503..12297)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"