LOCUS       62056_1116        8687 bp DNA     circular SYN 26-AUG-2019
DEFINITION  Cloning vector GWpNF, complete sequence.
ACCESSION   MK450605
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8687)
  AUTHORS   Siqueira Reis R.
  TITLE     Gateway(R) sites followed by nano and firefly (nLuc/Fluc) dual 
            luciferase for protoplast transformation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8687)
  AUTHORS   Siqueira Reis R.
  TITLE     Direct Submission
  JOURNAL   Submitted (26-JAN-2019) Department of Plant Molecular Biology, 
            University of Lausanne, Quartier UNIL-Sorge, Batiment Biophore, 
            Lausanne, Vaud 1015, Switzerland
REFERENCE   3  (bases 1 to 8687)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (26-JAN-2019) Department of Plant Molecular Biology, University of 
            Lausanne, Quartier UNIL-Sorge, Batiment Biophore, Lausanne, Vaud 
            1015, Switzerland"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..8687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      103..189
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      281..308
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     protein_bind    368..492
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        517..547
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             601..1257
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             1602..1904
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(1948..2072)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2086..2151
                     /codon_start=1
                     /label=F2A
                     /note="2A peptide from foot-and-mouth disease virus
                     polyprotein"
                     /translation="VKQLLNFDLLKLAGDVESNPGP"
     CDS             2170..2682
                     /codon_start=1
                     /label=Nluc
                     /note="NanoLuc(R) luciferase"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             2683..2709
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     terminator      2732..2980
                     /label=HSP terminator
                     /note="efficient transcription terminator from the
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     protein_bind    complement(2989..3013)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4748..6397
                     /codon_start=1
                     /label=luciferase
                     /note="firefly luciferase"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     terminator      6427..6679
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     promoter        7016..7086
                     /label=AmpR promoter
     CDS             7087..7944
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      8037..8625
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagca tggatctcgg
       61 ggacgtctaa ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct
      121 cagtcggaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      181 aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg
      241 gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat
      301 ggcctttttg cgtttctacc attcagctat gacatgatta cgaattcctc cccgagctcg
      361 aattatcaca agtttgtaca aaaaagctga acgagaaacg taaaatgata taaatatcaa
      421 tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat
      481 ccagtcacta tggcggccgc attaggcacc ccaggcttta cactttatgc ttccggctcg
      541 tataatgtgt ggattttgag ttaggatccg gcgagatttt caggagctaa ggaagctaaa
      601 atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa
      661 cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat
      721 attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt
      781 cacattcttg cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt
      841 gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa
      901 acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat
      961 tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag
     1021 aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg
     1081 gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc
     1141 gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtctgtga tggcttccat
     1201 gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa
     1261 acgcgtggat ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt
     1321 tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtgtgcta
     1381 tgaagcagcg tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata
     1441 tgatgtcaat atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg
     1501 tgccgaacgc tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga
     1561 aatgaacggc tcttttgctg acgagaacag ggactggtga aatgcagttt aaggtttaca
     1621 cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca
     1681 cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct
     1741 cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg
     1801 atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg
     1861 aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc
     1921 ttatacacag ccagtctgca ggtcgaccat agtgactgga tatgttgtgt tttacagtat
     1981 tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac
     2041 gtttctcgtt cagctttctt gtacaaagtg gtgatagctt ggccggtgaa acagctgttg
     2101 aattttgacc ttcttaagct tgcgggagac gtcgagtcca accccgggcc ctcgatcgag
     2161 gcacgggcaa tggtcttcac actcgaagat ttcgttgggg actggcgaca gacagccggc
     2221 tacaacctgg accaagtcct tgaacaggga ggtgtgtcca gtttgtttca gaatctcggg
     2281 gtgtccgtaa ctccgatcca aaggattgtc ctgagcggtg aaaatgggct gaagatcgac
     2341 atccatgtca tcatcccgta tgaaggtctg agcggcgacc aaatgggcca gatcgaaaaa
     2401 atttttaagg tggtgtaccc tgtggatgat catcacttta aggtgatcct gcactatggc
     2461 acactggtaa tcgacggggt tacgccgaac atgatcgact atttcggacg gccgtatgaa
     2521 ggcatcgccg tgttcgacgg caaaaagatc actgtaacag ggaccctgtg gaacggcaac
     2581 aaaattatcg acgagcgcct gatcaacccc gacggctccc tgctgttccg agtaaccatc
     2641 aacggagtga ccggctggcg gctgtgcgaa cgcattctgg cgtacccata cgatgttcca
     2701 gattacgctt gaccttagta aaagggtggg atatgaagat gaagatgaaa tatttggtgt
     2761 gtcaaataaa aagcttgtgt gcttaagttt gtgttttttt cttggcttgt tgtgttatga
     2821 atttgtggct ttttctaata ttaaatgaat gtaagatctc attataatga ataaacaaat
     2881 gtttctataa tccattgtga atgttttgtt ggatctcttc tgcagcatat aactactgta
     2941 tgtgctatgg tatggactat ggaatatgat taaagataag gcgcgccgac ccagctttct
     3001 tgtacaaagt ggtgatatca ctagtgtatg taggtatagt aacatgaaca agaacagact
     3061 caagtacaag atcgccaggg atccctcaac ttttgattcg ctatttgcag tgcacctgtg
     3121 gcgttcatca catcttttgt gacactgttt gcactggtca ttgctattac aaaggacctt
     3181 cctgatgttg aaggagatcg aaagtaagta actgcacgca taaccatttt ctttccgctc
     3241 tttggctcaa tccatttgac agtcaaagac aatgtttaac cagctccgtt tgatatattg
     3301 tctttatgtg tttgttcaag catgtttagt taatcatgcc tttgattgat cttgaatagg
     3361 ttccaaatat caaccctggc aacaaaactt ggagtgagaa acattgcatt cctcggttct
     3421 ggacttctgc tagtaaatta tgtttcagcc atatcactag ctttctacat gcctcaggtg
     3481 aattcatcta tttccgtctt aactatttcg gttaattaaa gcacgaacac cattactgca
     3541 tgtagaagct tgataaacta tcgccaccaa tttatttttg ttgcgatatt gttactttcc
     3601 tcagtatgca gctttgaaaa gaccaaccct cttatccttt aacaatgaac aggtttttag
     3661 aggtagcttg atgattcctg cacatgtgat cttggcttca ggcttaattt tccaggtaaa
     3721 gcattatgag atactcttat atctcttaca tacttttgag ataatgcaca agaacttcat
     3781 aactatatgc tttagtttct gcatttgaca ctgccaaatt cattaatctc taatatcttt
     3841 gttgttgatc tttggtagac atgggtacta gaaaaagcaa actacaccaa ggtaaaatac
     3901 ttttgtacaa acataaactc gttatcacgg aacatcaatg gagtgtatat ctaacggagt
     3961 gtagaaacat ttgattattg caggaagcta tctcaggata ttatcggttt atatggaatc
     4021 tcttctacgc agagtatctg ttattcccct tcctctagct ttcaatttca tggtgaggat
     4081 atgcagtttt ctttgtatat cattcttctt cttctttgta gcttggagtc aaaatcggtt
     4141 ccttcatgta catacatcaa ggatatgtcc ttctgaattt ttatatcttg caataaaaat
     4201 gcttgtacca attgaaacac cagctttttg agttctatga tcactgactt ggttctaacc
     4261 aaaaaaaaaa aatgtttaat ttacatatct aaaagtaggt ttagggaaac ctaaacagta
     4321 aaatatttgt atattattcg aatttcactc atcataaaaa cttaaattgc accataaaat
     4381 tttgttttac tattaatgat gtaatttgtg taacttaaga taaaaataat attccgtaag
     4441 ttaaccggct aaaaccacgt ataaaccagg gaacctgtta aaccggttct ttactggata
     4501 aagaaatgaa agcccatgta gacagctcca ttagagccca aaccctaaat ttctcatcta
     4561 tataaaagga gtgacattag ggtttttgtt cgtcctctta aagcttctcg ttttctctgc
     4621 cgtctctctc attcgcgcga cgcaaacgat cttcaggtga tcttctttct ccaaatcctc
     4681 tctcataact ctgatttcgt acttgtgtat ttgagctcac gctctgtttc tctcaccaca
     4741 gcccaccatg gaagacgcca aaaacataaa gaaaggcccg gcgccattct atccgctgga
     4801 agatggaacc gctggagagc aactgcataa ggctatgaag agatacgccc tggttcctgg
     4861 aacaattgct tttacagatg cacatatcga ggtggacatc acttacgctg agtacttcga
     4921 aatgtccgtt cggttggcag aagctatgaa acgatatggg ctgaatacaa atcacagaat
     4981 cgtcgtatgc agtgaaaact ctcttcaatt ctttatgccg gtgttgggcg cgttatttat
     5041 cggagttgca gttgcgcccg cgaacgacat ttataatgaa cgtgaattgc tcaacagtat
     5101 gggcatttcg cagcctaccg tggtgttcgt ttccaaaaag gggttgcaaa aaattttgaa
     5161 cgtgcaaaaa aagctcccaa tcatccaaaa aattattatc atggattcta aaacggatta
     5221 ccagggattt cagtcgatgt acacgttcgt cacatctcat ctacctcccg gttttaatga
     5281 atacgatttt gtgccagagt ccttcgatag ggacaagaca attgcactga tcatgaactc
     5341 ctctggatct actggtctgc ctaaaggtgt cgctctgcct catagaactg cctgcgtgag
     5401 attctcgcat gccagagatc ctatttttgg caatcaaatc attccggata ctgcgatttt
     5461 aagtgttgtt ccattccatc acggttttgg aatgtttact acactcggat atttgatatg
     5521 tggatttcga gtcgtcttaa tgtatagatt tgaagaagag ctgtttctga ggagccttca
     5581 ggattacaag attcaaagtg cgctgctggt gccaacccta ttctccttct tcgccaaaag
     5641 cactctgatt gacaaatacg atttatctaa tttacacgaa attgcttctg gtggcgctcc
     5701 cctctctaag gaagtcgggg aagcggttgc caagaggttc catctgccag gtatcaggca
     5761 aggatatggg ctcactgaga ctacatcagc tattctgatt acacccgagg gggatgataa
     5821 accgggcgcg gtcggtaaag ttgttccatt ttttgaagcg aaggttgtgg atctggatac
     5881 cgggaaaacg ctgggcgtta atcaaagagg cgaactgtgt gtgagaggtc ctatgattat
     5941 gtccggttat gtaaacaatc cggaagcgac caacgccttg attgacaagg atggatggct
     6001 acattctgga gacatagctt actgggacga agacgaacac ttcttcatcg ttgaccgcct
     6061 gaagtctctg attaagtaca aaggctatca ggtggctccc gctgaattgg aatccatctt
     6121 gctccaacac cccaacatct tcgacgcagg tgtcgcaggt cttcccgacg atgacgccgg
     6181 tgaacttccc gccgccgttg ttgttttgga gcacggaaag acgatgacgg aaaaagagat
     6241 cgtggattac gtcgccagtc aagtaacaac cgcgaaaaag ttgcgcggag gagttgtgtt
     6301 tgtggacgaa gtaccgaaag gtcttaccgg aaaactcgac gcaagaaaaa tcagagagat
     6361 cctcataaag gccaagaagg gcggaaagat cgccgtgtaa tgagcttgga atggatcttc
     6421 gatcccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt
     6481 cttgcgacga ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg
     6541 taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt
     6601 taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg
     6661 tcatctatgt tactagatcg ggaattgcca agctaattct tgaagacgaa agggcctcgt
     6721 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     6781 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     6841 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatggg accgactcgc
     6901 gctgtcagga gcatgccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata
     6961 tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttttca
     7021 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     7081 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     7141 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     7201 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     7261 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     7321 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     7381 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     7441 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     7501 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     7561 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     7621 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     7681 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     7741 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     7801 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     7861 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     7921 ggtgcctcac tgattaagca ttggtaatca gaattggtta attggttgta acattattca
     7981 gattgggccc cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     8041 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     8101 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     8161 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     8221 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     8281 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     8341 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     8401 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     8461 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     8521 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     8581 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     8641 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtt
//
