LOCUS       40924_35099        6753 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pPLV04_v2, complete sequence.
ACCESSION   .
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)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6753)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods 
            Mol. Biol. 1284, 421-431 (2015)"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (25-JUN-2014) Laboratory of Biochemistry, Wageningen University, 
            Dreijenlaan 3, Wageningen 6703HA, The Netherlands"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6753
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(66..654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(828..1640)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      1931..2366
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    2493..2515
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA
                     (truncated)"
     terminator      complement(2539..2791)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(2834..3625)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     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             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
                     /label=EGFP
                     /note="enhanced GFP"
     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"
     terminator      6434..6682
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_feature    6704..6728
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
ORIGIN
        1 gatctcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
       61 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      121 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
      181 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
      241 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
      301 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
      361 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
      421 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
      481 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
      541 gttaccttcg gaagaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
      601 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
      661 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
      721 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
      781 tttaaatcaa tctaaagtat atatgtgtaa cattggtcta gtgattagaa aaactcatcg
      841 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
      901 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
      961 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     1021 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     1081 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     1141 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     1201 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     1261 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     1321 aatgctgttt tccctgggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     1381 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     1441 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     1501 ggcttcccat acaatcggta gattgtcgca cctgattgcc cgacattatc gcgagcccat
     1561 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggccttga gcaagacgtt
     1621 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     1681 attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca
     1741 acgtggcttt gttgaataaa tcgaactttt gctgagttga aggatcagat cacgcatctt
     1801 cccgacaacg cagaccgttc cgtggcaaag caaaagttca aaatcaccaa ctggtccacc
     1861 tacaacaaag ctctcatcaa ccgtggctcc ctcactttct ggctggatga tggggcgatt
     1921 caggcgatcc ccatccaaca gcccgccgtc gagcgggctt ttttatcccc ggaagcctgt
     1981 ggatagaggg tagttatcca cgtgaaaccg ctaatgcccc gcaaagcctt gattcacggg
     2041 gctttccggc ccgctccaaa aactatccac gtgaaatcgc taatcagggt acgtgaaatc
     2101 gctaatcgga gtacgtgaaa tcgctaataa ggtcacgtga aatcgctaat caaaaaggca
     2161 cgtgagaacg ctaatagccc tttcagatca acagcttgca aacacccctc gctccggcaa
     2221 gtagttacag caagtagtat gttcaattag cttttcaatt atgaatatat atatcaatta
     2281 ttggtcgccc ttggcttgtg gacaatgcgc tacgcgcacc ggctccgccc gtggacaacc
     2341 gcaagcggtt gcccaccgtc gagcgccttt gcccacaacc cggcggccgg ccgcaacaga
     2401 tcgttttata aatttttttt tttgaaaaag aaaaagcccg aaaggcggca acctctcggg
     2461 cttctggatt tccgatcccc ggaattagat cttggcagga tatattgtgg tgtaacgtta
     2521 tcagcttgca tgccggtcga tctagtaaca tagatgacac cgcgcgcgat aatttatcct
     2581 agtttgcgcg ctatattttg ttttctatcg cgtattaaat gtataattgc gggactctaa
     2641 tcataaaaac ccatctcata aataacgtca tgcattacat gttaattatt acatgcttaa
     2701 cgtaattcaa cagaaattat atgataatca tcgcaagacc ggcaacagga ttcaatctta
     2761 agaaacttta ttgccaaatg tttgaacgat ctgcttgact ctagctagag tccgaacccc
     2821 agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg
     2881 gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag
     2941 caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac
     3001 agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc
     3061 cgtgggtcac gacgagatcc tcgccgtcgg gcatccgcgc cttgagcctg gcgaacagtt
     3121 cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt
     3181 ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag
     3241 ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag
     3301 gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc
     3361 ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc
     3421 acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga
     3481 caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga
     3541 ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg
     3601 cgtgcaatcc atcttgttca atcatgcctc gatcgagttg agagtgaata tgagactcta
     3661 attggatacc gaggggaatt tatggaacgt cagtggagca tttttgacaa gaaatatttg
     3721 ctagctgata gtgaccttag gcgacttttg aacgcgcaat aatggtttct gacgtatgtg
     3781 cttagctcat taaactccag aaacccgcgg ctcagtggct ccttcaacgt tgcggttctg
     3841 tcagttccaa acgtaaaacg gcttgtcccg cgtcatcggc gggggtcata acgtgactcc
     3901 cttaattctc cgctcatgat cgataacatt aacgtttaca atttccattc gccattcagg
     3961 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     4021 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     4081 cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg cgaattgggt
     4141 accgggcccc ccctcgaggt cgacggtatc gataagcttg atatcgaatt ctagttggaa
     4201 tgggttaacc caactccata aggatccatg gctccaaaga agaagagaaa ggtcatggtg
     4261 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     4321 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     4381 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     4441 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     4501 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     4561 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     4621 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     4681 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     4741 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     4801 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     4861 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     4921 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gagatccatg
     4981 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     5041 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     5101 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     5161 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     5221 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     5281 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     5341 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     5401 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     5461 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     5521 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     5581 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     5641 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagagatcc
     5701 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     5761 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     5821 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     5881 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     5941 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     6001 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     6061 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     6121 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     6181 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     6241 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     6301 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     6361 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     6421 agatccgatc cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt
     6481 tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat
     6541 taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt
     6601 atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg
     6661 cgcggtgtca tctatgtcta gagcggccgc cacgcgtagg ccttgacagg atatattggc
     6721 gggtaaacta agtcgctgta tgtgtttgtt tga
//
