LOCUS       Exported               11447 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway binary vector pGWB665 DNA, complete sequence.
ACCESSION   AP018991
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11447)
  AUTHORS   Momtaz S, Dutta A, Tanaka Y, Aboulela M, Nishimura K, Sugiura S, 
            Niwa T, Maeo K, Goto-Yamada S, Kimura T, Ishiguro S, Mano S, 
            Nakagawa T.
  TITLE     Gateway Binary Vectors with Organelle-Targeted Fluorescent Proteins 
            for Highly Sensitive Reporter Assay in Gene Expression Analysis of 
            Plants
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 11447)
  AUTHORS   Momtaz S, Dutta A, Tanaka Y, Aboulela M, Nishimura K, Sugiura S, 
            Niwa T, Maeo K, Goto-Yamada S, Kimura T, Ishiguro S, Mano S, 
            Nakagawa T.
  TITLE     Direct Submission
  JOURNAL   Submitted (17-OCT-2018) Contact:Tsuyoshi Nakagawa Shimane 
            University, Interdisciplinary Center for Science Research; 1060 
            Nishikawatsu, Matsue, Shimane 690-8504, Japan
REFERENCE   3  (bases 1 to 11447)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     constructed using pPZP221.
FEATURES             Location/Qualifiers
     source          1..11447
                     /organism="Gateway binary vector pGWB665"
                     /mol_type="other DNA"
                     /db_xref="taxon:2479752"
     misc_feature    complement(54..78)
                     /label=T-DNA right border
                     /note="T-DNA right border"
     primer_bind     281..297
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    complement(338..462)
                     /label=attR1
                     /note="attR1"
     protein_bind    338..462
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        499..529
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             583..1242
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=cat
                     /protein_id="BBG75631.1"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     gene            583..1242
                     /gene="cat"
                     /label=cat
     CDS             1584..1889
                     /codon_start=1
                     /gene="ccdB"
                     /label=ccdB
                     /protein_id="BBG75632.1"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     gene            1584..1889
                     /gene="ccdB"
                     /label=ccdB
     misc_feature    1930..2054
                     /label=attR2
                     /note="attR2"
     protein_bind    complement(1930..2054)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2065..2811
                     /codon_start=1
                     /gene="nls-sgfp"
                     /product="nucleus-targeted synthetic green fluorescent 
                     protein"
                     /label=nls-sgfp
                     /protein_id="BBG75633.1"
                     /translation="MAPKKKRKVMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEG
                     DATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQ
                     ERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIM
                     ADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN
                     EKRDHMVLLEFVTAAGITHGMDELYK"
     gene            2065..2811
                     /gene="nls-sgfp"
                     /label=nls-sgfp
     CDS             2071..2091
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2092..2811
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     regulatory      2817..3226
                     /regulatory_class="terminator"
                     /note="NOS terminator"
     terminator      2833..3085
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(3119..3135)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3143..3159
                     /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)."
     promoter        complement(3167..3197)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3212..3233
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     terminator      3269..3521
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     regulatory      complement(3271..3537)
                     /regulatory_class="terminator"
                     /note="NOS terminator"
     CDS             complement(3681..4232)
                     /codon_start=1
                     /gene="bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=bar
                     /protein_id="BBG75634.1"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     gene            complement(3681..4232)
                     /gene="bar"
                     /label=bar
     regulatory      complement(4260..4546)
                     /regulatory_class="promoter"
                     /note="NOS promoter"
     promoter        complement(4284..4463)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     misc_feature    complement(4969..4993)
                     /label=T-DNA left border
                     /note="T-DNA left border"
     CDS             5514..6305
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      6551..7139
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    7325..7465
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      7809..8003
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(8069..9142)
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     CDS             complement(9571..10200)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"