LOCUS       Exported                4971 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Binary vector pCLEAN-G146, complete sequence.
ACCESSION   EU186088
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4971)
  AUTHORS   Thole V, Worland B, Snape JW, Vain P.
  TITLE     The pCLEAN dual binary vector system for Agrobacterium-mediated 
            plant transformation
  JOURNAL   Plant Physiol. 145 (4), 1211-1219 (2007)
  PUBMED    17932303
REFERENCE   2  (bases 1 to 4971)
  AUTHORS   Thole V, Vain P.
  TITLE     Direct Submission
  JOURNAL   Submitted (01-OCT-2007) Department of Crop Genetics, John Innes 
            Centre, Colney Lane, Norwich NR4 7UH, UK
REFERENCE   3  (bases 1 to 4971)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4971
                     /organism="Binary vector pCLEAN-G146"
                     /mol_type="other DNA"
                     /db_xref="taxon:478691"
     misc_feature    1..2481
                     /note="Cloning vector pGreenII0000 (Genbank Accession 
                     Number EF590266)-based backbone sequence"
     rep_origin      1..720
                     /label=pUC-ori
                     /note="pUC-ori"
     rep_origin      complement(61..649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(820..1635)
                     /codon_start=1
                     /gene="nptI"
                     /product="NptI"
                     /label=nptI
                     /note="kanamycin resistance"
                     /protein_id="ABW84176.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANVVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            complement(820..1635)
                     /gene="nptI"
                     /label=nptI
     rep_origin      1921..2473
                     /label=pSa-ori
                     /note="pSa-ori"
     rep_origin      1926..2361
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    2482..4971
                     /note="Basic pCLEAN T-DNA region no. 2 (Thole et al., 2007;
                     http://www.jic.ac.uk/staff/philippe-vain/vectors.htm) 
                     including a modified pGreenII0000 T-DNA and containing 
                     additional restriction sites outside the T-DNA left border 
                     sequence"
     misc_feature    complement(2538..4100)
                     /note="CaMV35S:mGFP5-ER:SPA expression unit inserted in 
                     vector backbone sequence"
     regulatory      complement(2538..2834)
                     /regulatory_class="terminator"
                     /note="soybean-derived poly(A)signal"
     CDS             complement(2855..3646)
                     /codon_start=1
                     /gene="mGFP5-ER"
                     /product="mGFP5-ER"
                     /label=mGFP5-ER
                     /note="modified green fluorescent protein mGFP5-ER gene 
                     (Haseloff et al. (1997) Proc Natl Acad Sci USA 94: 
                     2122-2127; Genbank Accession Number U87974) containing one 
                     nucleotide variation resulting in an amino acid change at 
                     position 46 (His[GTG] to Tyr [GTA])"
                     /protein_id="ABW84177.1"
                     /translation="MKTNLFLFLIFSLLLSLSSAEFSKGEELFTGVVPILVELDGDVNG
                     YKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDF
                     FKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLE
                     YNYNSHNVYIMADKQKNGIKANFKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYL
                     STQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKHDEL"
     gene            complement(2855..3646)
                     /gene="mGFP5-ER"
                     /label=mGFP5-ER
     CDS             complement(2870..3580)
                     /codon_start=1
                     /gene="mgfp5"
                     /product="GFP with folding enhancement mutations"
                     /label=mgfp5
                     /note="suitable for expression in plants due to removal of 
                     a cryptic intron"
                     /translation="SKGEELFTGVVPILVELDGDVNGYKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKAN
                     FKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     regulatory      complement(3673..4100)
                     /regulatory_class="promoter"
                     /note="35S promoter of CaMV"
     promoter        complement(3679..4023)
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     misc_feature    4141..4165
                     /note="25-nt long consensus T-DNA left border sequence of 
                     the nopaline vector pTiT37 (Slightom et al., (1985) EMBO J 
                     4:3069-3077)"
     misc_feature    4166..4910
                     /note="745-nt long inner T-DNA region based on T-DNA region
                     (Genbank Accession Number EU048862; Hellens et al. (2000) 
                     Plant Mol Biol 42: 819-832) of cloning vector pGreenII0000"
     CDS             complement(4189..4554)
                     /codon_start=1
                     /gene="lacZ"
                     /product="LacZ"
                     /label=lacZ
                     /protein_id="ABW84178.1"
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     gene            complement(4189..4554)
                     /gene="lacZ"
                     /label=lacZ
     primer_bind     4338..4354
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4364..4382
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    4391..4498
                     /label=multiple cloning site
                     /note="multiple cloning site"
     primer_bind     4408..4424
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4458..4474)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(4511..4529)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4550..4566)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4574..4590
                     /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(4598..4628)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4643..4664
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    4911..4935
                     /note="25-nt long consensus T-DNA right border sequence of 
                     the nopaline vector pTiT37 (Slightom et al., (1985) EMBO J 
                     4:3069-3077)"
     misc_feature    4936..4949
                     /note="right border overdrive sequence (Peralta et al. 
                     (1986) EMBO J 5: 1137-1142)"