LOCUS       Exported                4347 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Binary vector pCLEAN-G182, complete sequence.
ACCESSION   EU186092
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4347)
  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 4347)
  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 4347)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4347
                     /organism="Binary vector pCLEAN-G182"
                     /mol_type="other DNA"
                     /db_xref="taxon:478694"
     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="ABW84186.1"
                     /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    complement(2536..4098)
                     /note="CaMV35S:mGFP5-ER:SPA expression unit inserted in 
                     vector backbone sequence outside the left border sequence 
                     of basic pCLEAN T-DNA region no. 5 (Thole et al., 2007; 
                     http://www.jic.ac.uk/staff/philippe-vain/vectors.htm)"
     regulatory      complement(2536..2832)
                     /regulatory_class="terminator"
                     /note="soybean-derived poly(A)signal"
     CDS             complement(2853..3644)
                     /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="ABW84187.1"
                     /translation="MKTNLFLFLIFSLLLSLSSAEFSKGEELFTGVVPILVELDGDVNG
                     YKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDF
                     FKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLE
                     YNYNSHNVYIMADKQKNGIKANFKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYL
                     STQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKHDEL"
     gene            complement(2853..3644)
                     /gene="mGFP5-ER"
                     /label=mGFP5-ER
     CDS             complement(2868..3578)
                     /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(3671..4098)
                     /regulatory_class="promoter"
                     /note="35S promoter of CaMV"
     promoter        complement(3677..4021)
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     misc_feature    4131..4347
                     /note="Basic pCLEAN T-DNA region no. 5; outer left border 
                     sequence has been used to insert CaMV35S:mGFP5-ER:SPA 
                     expression unit"
     misc_feature    4137..4161
                     /note="T-DNA left border No. 1 sequence belonging to a 
                     double left border sequence separated by a 26-nt long 
                     spacer sequence from left border No. 2 sequence (Thole et 
                     al., 2007); left border No. 1 is a 25-nt long consensus 
                     sequence of the nopaline vector pTiT37 (Slightom et al., 
                     (1985) EMBO J 4:3069-3077)"
     misc_feature    4162..4187
                     /note="26-nt long spacer sequence separating left border 
                     No. 1 and No. 2 of the double left border sequence (Thole 
                     et al., 2007)"
     misc_feature    4188..4212
                     /note="T-DNA left border No. 2 sequence belonging to a 
                     double left border sequence separated by a 26-nt long 
                     spacer sequence from left border No. 1 sequence (Thole et 
                     al., 2007); left border No. 2 is a 25-nt long consensus 
                     sequence of the nopaline vector pTiT37 (Slightom et al., 
                     (1985) EMBO J 4:3069-3077)"
     misc_feature    4213..4264
                     /note="Inner T-DNA region containing 52 nt-long multiple 
                     cloning site with NotI, PacI, SfiI, BsiWI, ApaI, XhoI and 
                     SalI restriction sites"
     misc_feature    4265..4289
                     /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    4290..4313
                     /note="right border overdrive sequence (Peralta et al. 
                     (1986) EMBO J 5: 1137-1142)"