LOCUS       40924_36588        8740 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-06 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8740)
  AUTHORS   Saisho T, Shibahara K, Nakao A, Tanaka Y, Nishimura K, Kimura T, 
            Nakagawa T.
  TITLE     Improved Gateway Recycling Cloning System for Multigene Construction
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8740)
  AUTHORS   Saisho T, Shibahara K, Nakao A, Tanaka Y, Nishimura K, Kimura T, 
            Nakagawa T.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-NOV-2015) Contact:Tsuyoshi Nakagawa Shimane 
            University, Interdisciplinary Center for Science Research; 1060 
            Nishikawatsu, Matsue, Shimane 690-8504, Japan
REFERENCE   3  (bases 1 to 8740)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8740)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (02-NOV-2015) Contact:Tsuyoshi Nakagawa Shimane University, 
            Interdisciplinary Center for Science Research; 1060 Nishikawatsu, 
            Matsue, Shimane 690-8504, Japan"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     constructed using pDONR201.
FEATURES             Location/Qualifiers
     source          1..8740
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    28..127
                     /label=attL1
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    674..1071
                     /label=MD8
                     /note="MD8"
     promoter        1192..1375
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     CDS             1393..2106
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELY"
     protein_bind    2110..2234
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2271..2301
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             2355..3011
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             3356..3658
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3702..3826)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      3864..4116
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4325..4449
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4474..4504
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4776..4816
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4908..5765)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5766..5870)
                     /label=AmpR promoter
     protein_bind    complement(6167..6290)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6616..6711)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6835..7641
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7787..8375
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8510..8537)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8629..8715)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
ORIGIN
        1 gttaacgcta gcatggatct cgggccccaa ataatgattt tattttgact gatagtgacc
       61 tgttcgttgc aacaaattga tgagcaatgc ttttttataa tgccaacttt gtatacaaaa
      121 gtaggctgga tgaatcgaat aacttcttct tcaaatgcgc aaacgaagaa atttcctcag
      181 tctcagccac agccgccgga gaaaaatgag aaaaaactga aaagggagag atttccggtg
      241 aaccctttga gagatttcag tacaagaagc tctggaagca gcagttgtag caatgagagt
      301 gcttctggtt caacttccgg tgaagcatcc aacggttgtc acaggttctt gctctctcat
      361 tctttctcat cgtcttcttc atcatcttct tcttcgttgg gagtttttcc tcgtagacca
      421 gtgagatcag tagctaaaaa ccccaaatct gctccggtag ttagtaaacc cttaattaga
      481 aagaaaccaa gctctcttga ggaggttaag ttgaaatcta ctcttactga gaaacctaat
      541 ttgctgaagt ctcagagatg caagacaaat cctgtatctg gaaagagacc aacttgtaag
      601 attaccatga aaccggagaa agtttcggtt ttgaagaaac aatcttctgt ttctaggtaa
      661 gctgcagtgt tttcatcaca cgtaatatca tccattttat attatgatcc acaaacagca
      721 aaggcccatt aatctcatta tattttagat taattgattg caattgccca acttctccac
      781 tcagcaatgt tagttttaaa gacacatcaa gttagtgtat tttattttta atctttctat
      841 aaaaaacaac actttgtttt tgtctatttc ttattctgtg aaaaagagaa atgaaaaagt
      901 aaaaaagata acaaaaaggc tggtttttca cggtgagaat ttttcatggt cacgcaataa
      961 cttaaatatg aactattaaa ataactaaga ttgaaaaatt agcatgatca aaagaagaag
     1021 aaaaaaaaga caacatgaag cggaaaggaa aaaaaagaaa gctgaaaatc taagcttatc
     1081 atgagcggag aattaaggga gtcacgttat gacccccgcc gatgacgcgg gacaagccgt
     1141 tttacgtttg gaactgacag aaccgcaacg ttgaaggagc cactcagccg cgggtttctg
     1201 gagtttaatg agctaagcac atacgtcaga aaccattatt gcgcgttcaa aagtcgccta
     1261 aggtcactat cagctagcaa atatttcttg tcaaaaatgc tccactgacg ttccataaat
     1321 tcccctcggt atccaattag agtctcatat tcactctcaa tccaaataat ctgcaccgaa
     1381 tctagaggat ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1441 gtcgagctgg acggcgacgt gaacggccac aagttcagcg tgtccggcga gggcgagggc
     1501 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1561 ccctggccca ccctcgtgac caccttcacc tacggcgtgc agtgcttcag ccgctacccc
     1621 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1681 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1741 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1801 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1861 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1921 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1981 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     2041 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactcacgg catggacgag
     2101 ctgtacatca caagtttgta caaaaaagct gaacgagaaa cgtaaaatga tataaatatc
     2161 aatatattaa attagatttt gcataaaaaa cagactacat aatactgtaa aacacaacat
     2221 atccagtcac tatggcggcc cctcgagggg ccgcattagg caccccaggc tttacacttt
     2281 atgcttccgg ctcgtataat gtgtggattt tgagttagga tccggcgaga ttttcaggag
     2341 ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat
     2401 ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga
     2461 ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt
     2521 atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg
     2581 caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc
     2641 atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt
     2701 ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta
     2761 aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt
     2821 ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat
     2881 attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgtct
     2941 gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc
     3001 agggcggggc gtaaacgcgt ggatccggct tactaaaagc cagataacag tatgcgtatt
     3061 tgcgcgctga tttttgcggt ataagaatat atactgatat gtatacccga agtatgtcaa
     3121 aaagaggtgt gctatgaagc agcgtattac agtgacagtt gacagcgaca gctatcagtt
     3181 gctcaaggca tatatgatgt caatatctcc ggtctggtaa gcacaaccat gcagaatgaa
     3241 gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc aggaagggat ggctgaggtc
     3301 gcccggttta ttgaaatgaa cggctctttt gctgacgaga acagggactg gtgaaatgca
     3361 gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag
     3421 tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct
     3481 gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg
     3541 catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga
     3601 tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata
     3661 aatgtcaggc tcccttatac acagccagtc tgcaggtcga ccatagtgac tggatatgtt
     3721 gtgttttaca gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat
     3781 ttatatcatt ttacgtttct cgttcagctt tcttgtacaa agtggtgatg tacaagtaaa
     3841 gcggcccgag ctcgaatttc cccgatcgtt caaacatttg gcaataaagt ttcttaagat
     3901 tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc
     3961 atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag
     4021 tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata
     4081 aattatcgcg cgcggtgtca tctatgttac tagatcggga attctagctt cgacaattat
     4141 tttggactct catgttctca agctgaaatc caaaagaaac gtgatgttgg ttcagatttg
     4201 gaatcactga caatgaagtt tcagtcagcc ggtttgtcac caaggatcca ggcttcatca
     4261 tgggagccga gccgttcaag tttcaacttc gactatttgg atccgtccta gtggatcccc
     4321 catcacaact ttgtatagaa aagttgaacg agaaacgtaa aatgatataa atatcaatat
     4381 attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca
     4441 gtcactatgg cggccgcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     4501 aatgtgtgga ttttgagtta ggatccggcg agattttcag gagctaagga agctaaaatg
     4561 gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat
     4621 tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt
     4681 acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac
     4741 attcttgccc gcctgatgaa tgctcatccg gaattgcgat cgcatttaaa tggccggccc
     4801 ttaattaagg cgcgccgttt aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4861 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4921 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4981 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5041 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5101 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5161 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5221 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5281 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5341 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5401 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5461 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5521 acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5581 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5641 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5701 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5761 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5821 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5881 cgaaaagtgc cacctgaaac gggcgacgga tggtgatccc cctggccagt gcacgtctgc
     5941 tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc
     6001 gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg
     6061 atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat
     6121 aaatgtcagg ctcccttata cacagccagt ctgcaggtcg atcgaccata gtgactggat
     6181 atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt
     6241 gatatttata tcattttacg tttctcgttc aactttatta tacaaagttg tgatccacat
     6301 cttctgattc tattgatctc tcacagtttc agagaggttt agttgacagg agttcttgtc
     6361 atcctcatgt aacactggat aaagtgtcaa ggacacattt gagagtagag caaacaaata
     6421 gccacatgcc tgaaatgaaa tcccaacaga ttacagatac tgaatttgat attcaaaatc
     6481 acaaagaaag tgctgcttct ctgggaaaag gaagagaatt gcttccatgc tctgctgcag
     6541 agtcaataag cactgatgga ggtggtctga tttgttctga ggattcaaat tggatggcat
     6601 gttacaagaa ttacccaact ttttaataca aagttggcat tataagaaag cattgcttat
     6661 caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt tgccatccag
     6721 ctgcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata
     6781 tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     6841 catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat
     6901 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
     6961 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     7021 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     7081 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa
     7141 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     7201 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     7261 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     7321 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     7381 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     7441 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     7501 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     7561 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     7621 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc
     7681 attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagtttt
     7741 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     7801 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     7861 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     7921 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     7981 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     8041 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     8101 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     8161 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     8221 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     8281 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     8341 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     8401 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     8461 ctgtggataa ccgtattacc gctagccagg aagagtttgt agaaacgcaa aaaggccatc
     8521 cgtcaggatg gccttctgct tagtttgatg cctggcagtt tatggcgggc gtcctgcccg
     8581 ccaccctccg ggccgttgct tcacaacgtt caaatccgct cccggcggat ttgtcctact
     8641 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt ccgactgagc
     8701 ctttcgtttt atttgatgcc tggcagttcc ctactctcgc
//
