LOCUS       Exported                6753 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pPLV04_v2, complete sequence.
ACCESSION   KM062047
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6753)
  AUTHORS   Wendrich JR, Liao CY, van den Berg WA, De Rybel B, Weijers D.
  TITLE     Ligation-independent cloning for plant research
  JOURNAL   Methods Mol. Biol. 1284, 421-431 (2015)
  PUBMED    25757785
REFERENCE   2  (bases 1 to 6753)
  AUTHORS   Wendrich JR, Liao C-Y., van den Berg WAM., De Rybel B, Weijers D.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-JUN-2014) Laboratory of Biochemistry, Wageningen 
            University, Dreijenlaan 3, Wageningen 6703HA, The Netherlands
REFERENCE   3  (bases 1 to 6753)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6753
                     /organism="Cloning vector pPLV04_v2"
                     /mol_type="other DNA"
                     /db_xref="taxon:1548278"
     rep_origin      complement(66..654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      complement(719..6742)
                     /direction=LEFT
                     /note="ColE; high copy number E. coli origin of 
                     replication"
     CDS             complement(825..1640)
                     /codon_start=1
                     /product="nptI"
                     /label=nptI
                     /note="kanamycin resistance gene"
                     /protein_id="AIQ78252.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      1931..2366
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     rep_origin      1985..2395
                     /note="pSa ORI; pSa origin or replication"
     misc_feature    2493..2519
                     /note="LB; T-DNA left border"
     misc_feature    2493..2515
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     regulatory      complement(2538..2782)
                     /regulatory_class="terminator"
                     /note="tNOS; nopaline synthase terminator (tNOS)"
     terminator      2539..2791
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(2831..3625)
                     /codon_start=1
                     /product="nptII"
                     /label=nptII
                     /note="kanamycin resistance gene"
                     /protein_id="AIQ78253.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     regulatory      complement(3627..3930)
                     /regulatory_class="promoter"
                     /note="pNOS; nopaline synthase (NOS) promoter"
     promoter        complement(3659..3838)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     4085..4101
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4111..4129
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4155..4171
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    4192..4221
                     /note="LIC; ligation-independent cloning (LIC) site"
     CDS             4228..6420
                     /codon_start=1
                     /product="n3xGFP"
                     /label=n3xGFP
                     /note="nuclear (SV40) 3x green fluorescent protein"
                     /protein_id="AIQ78254.1"
                     /translation="MAPKKKRKVMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEG
                     DATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQ
                     ERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIM
                     ADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN
                     EKRDHMVLLEFVTAAGITLGMDELYKRSMVSKGEELFTGVVPILVELDGDVNGHKFSVS
                     GEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMP
                     EGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSH
                     NVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSAL
                     SKDPNEKRDHMVLLEFVTAAGITLGMDELYKRSMVSKGEELFTGVVPILVELDGDVNGH
                     KFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFF
                     KSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY
                     NYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLS
                     TQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     CDS             4234..4254
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4255..4971
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             4978..5694
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             5701..6420
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     regulatory      6443..6682
                     /regulatory_class="terminator"
                     /note="tNOS; nopaline synthase terminator (tNOS)"
     misc_feature    6704..6752
                     /note="RB; T-DNA right border"
     misc_feature    6704..6728
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"