LOCUS       40924_36583        8747 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-05 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8747)
  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  JOURNAL   Unpublished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 8747)
  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 8747)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8747)
  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            "Unpublished"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..8747
                     /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"
     protein_bind    1394..1518
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1555..1585
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1639..2295
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2640..2942
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(2986..3110)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3127..3843
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     terminator      3871..4123
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4332..4456
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4481..4511
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4783..4823
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4915..5772)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5773..5877)
                     /label=AmpR promoter
     protein_bind    complement(6174..6297)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6623..6718)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6842..7648
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7794..8382
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8517..8544)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8636..8722)
                     /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 tctagagtta tcaacaagtt tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa
     1441 tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca
     1501 acatatccag tcactatggc ggcccctcga ggggccgcat taggcacccc aggctttaca
     1561 ctttatgctt ccggctcgta taatgtgtgg attttgagtt aggatccgtc gagattttca
     1621 ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc
     1681 caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac
     1741 cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag
     1801 ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt
     1861 atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt
     1921 ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg
     1981 cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc
     2041 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc
     2101 agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc
     2161 aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc
     2221 gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag
     2281 tggcagggcg gggcgtaatc tagaggatcc ggcttactaa aagccagata acagtatgcg
     2341 tatttgcgcg ctgatttttg cggtataaga atatatactg atatgtatac ccgaagtatg
     2401 tcaaaaagag gtatgctatg aagcagcgta ttacagtgac agttgacagc gacagctatc
     2461 agttgctcaa ggcatatatg atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa
     2521 tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag ggatggctga
     2581 ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac gagaacaggg gctggtgaaa
     2641 tgcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac
     2701 agagtgatat tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc
     2761 tgctgtcaga taaagtcccc cgtgaacttt acccggtggt gcatatcggg gatgaaagct
     2821 ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg
     2881 ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa
     2941 tataaatgtc aggctccctt atacacagcc agtctgcagg tcgaccatag tgactggata
     3001 tgttgtgttt tacagtatta tgtagtctgt tttttatgca aaatctaatt taatatattg
     3061 atatttatat cattttacgt ttctcgttca gctttcttgt acaaagtggt tcgatctaga
     3121 ggatccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     3181 ctggacggcg acgtgaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     3241 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     3301 cccaccctcg tgaccacctt cacctacggc gtgcagtgct tcagccgcta ccccgaccac
     3361 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     3421 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     3481 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     3541 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     3601 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     3661 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     3721 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     3781 atggtcctgc tggagttcgt gaccgccgcc gggatcactc acggcatgga cgagctgtac
     3841 aagtaaagcg gcccgagctc gaatttcccc gatcgttcaa acatttggca ataaagtttc
     3901 ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac
     3961 gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg
     4021 attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac
     4081 taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgggaatt ctagcttcga
     4141 caattatttt ggactctcat gttctcaagc tgaaatccaa aagaaacgtg atgttggttc
     4201 agatttggaa tcactgacaa tgaagtttca gtcagccggt ttgtcaccaa ggatccaggc
     4261 ttcatcatgg gagccgagcc gttcaagttt caacttcgac tatttggatc cgtcctagtg
     4321 gatcccccat cacaactttg tatagaaaag ttgaacgaga aacgtaaaat gatataaata
     4381 tcaatatatt aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac
     4441 atatccagtc actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg
     4501 ctcgtataat gtgtggattt tgagttagga tccggcgaga ttttcaggag ctaaggaagc
     4561 taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa
     4621 agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct
     4681 ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt
     4741 tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttgcgatcgc atttaaatgg
     4801 ccggccctta attaaggcgc gccgtttaag gatcttcacc tagatccttt taaattaaaa
     4861 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4921 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4981 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5041 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5101 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5161 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5221 cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5281 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5341 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5401 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5461 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5521 ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5581 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5641 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5701 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5761 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5821 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5881 atttccccga aaagtgccac ctgaaacggg cgacggatgg tgatccccct ggccagtgca
     5941 cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa
     6001 agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa
     6061 gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg
     6121 ggaatataaa tgtcaggctc ccttatacac agccagtctg caggtcgatc gaccatagtg
     6181 actggatatg ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta
     6241 atatattgat atttatatca ttttacgttt ctcgttcaac tttattatac aaagttgtga
     6301 tccacatctt ctgattctat tgatctctca cagtttcaga gaggtttagt tgacaggagt
     6361 tcttgtcatc ctcatgtaac actggataaa gtgtcaagga cacatttgag agtagagcaa
     6421 acaaatagcc acatgcctga aatgaaatcc caacagatta cagatactga atttgatatt
     6481 caaaatcaca aagaaagtgc tgcttctctg ggaaaaggaa gagaattgct tccatgctct
     6541 gctgcagagt caataagcac tgatggaggt ggtctgattt gttctgagga ttcaaattgg
     6601 atggcatgtt acaagaatta cccaactttt taatacaaag ttggcattat aagaaagcat
     6661 tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc
     6721 catccagctg cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa
     6781 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     6841 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
     6901 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     6961 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     7021 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     7081 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     7141 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     7201 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     7261 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     7321 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     7381 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     7441 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     7501 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     7561 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     7621 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg
     7681 gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt
     7741 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     7801 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     7861 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     7921 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     7981 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     8041 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     8101 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     8161 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     8221 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     8281 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     8341 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     8401 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     8461 cctgattctg tggataaccg tattaccgct agccaggaag agtttgtaga aacgcaaaaa
     8521 ggccatccgt caggatggcc ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc
     8581 ctgcccgcca ccctccgggc cgttgcttca caacgttcaa atccgctccc ggcggatttg
     8641 tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtcttccg
     8701 actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgc
//
