LOCUS       40924_36708        8731 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-61 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8731)
  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  JOURNAL   Unpublished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 8731)
  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 8731)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8731)
  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            "Unpublished"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..8731
                     /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..2103
                     /codon_start=1
                     /label=TagRFP
                     /note="monomeric derivative of red fluorescent protein from
                     Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     protein_bind    2113..2237
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2274..2304
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             2358..3014
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             3359..3661
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3705..3829)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      3855..4107
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4316..4440
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4465..4495
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4767..4807
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4899..5756)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5757..5861)
                     /label=AmpR promoter
     protein_bind    complement(6158..6281)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6607..6702)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6826..7632
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7778..8366
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8501..8528)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8620..8706)
                     /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 tctagaatga gcatggtgtc taagggcgaa gagctgatta aggagaacat gcacatgaag
     1441 ctgtacatgg agggcaccgt gaacaaccac cacttcaagt gcacatccga gggcgaaggc
     1501 aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc
     1561 ttcgccttcg acatcctggc taccagcttc atgtacggca gcagaacctt catcaaccac
     1621 acccagggca tccccgactt ctttaagcag tccttccctg agggcttcac atgggagaga
     1681 gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac
     1741 ggctgcctca tctacaacgt caagatcaga ggggtgaact tcccatccaa cggccctgtg
     1801 atgcagaaga aaacactcgg ctgggaggcc aacaccgaga tgctgtaccc cgctgacggc
     1861 ggcctggaag gcagaagcga catggccctg aagctcgtgg gcgggggcca cctgatctgc
     1921 aacttcaaga ccacatacag atccaagaaa cccgctaaga acctcaagat gcccggcgtc
     1981 tactatgtgg accacagact ggaaagaatc aaggaggccg acaaagagac ctacgtcgag
     2041 cagcacgagg tggctgtggc cagatactgc gacctcccta gcaaactggg gcacaaactt
     2101 aatgctgtta tcacaagttt gtacaaaaaa gctgaacgag aaacgtaaaa tgatataaat
     2161 atcaatatat taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa
     2221 catatccagt cactatggcg gcccctcgag gggccgcatt aggcacccca ggctttacac
     2281 tttatgcttc cggctcgtat aatgtgtgga ttttgagtta ggatccggcg agattttcag
     2341 gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc
     2401 aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc
     2461 agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt
     2521 tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta
     2581 tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt
     2641 tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc
     2701 agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc
     2761 ctaaagggtt tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca
     2821 gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca
     2881 aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg
     2941 tctgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt
     3001 ggcagggcgg ggcgtaaacg cgtggatccg gcttactaaa agccagataa cagtatgcgt
     3061 atttgcgcgc tgatttttgc ggtataagaa tatatactga tatgtatacc cgaagtatgt
     3121 caaaaagagg tgtgctatga agcagcgtat tacagtgaca gttgacagcg acagctatca
     3181 gttgctcaag gcatatatga tgtcaatatc tccggtctgg taagcacaac catgcagaat
     3241 gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa atcaggaagg gatggctgag
     3301 gtcgcccggt ttattgaaat gaacggctct tttgctgacg agaacaggga ctggtgaaat
     3361 gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca
     3421 gagtgatatt attgacacgc ccgggcgacg gatggtgatc cccctggcca gtgcacgtct
     3481 gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg
     3541 gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc
     3601 tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat
     3661 ataaatgtca ggctccctta tacacagcca gtctgcaggt cgaccatagt gactggatat
     3721 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga
     3781 tatttatatc attttacgtt tctcgttcag ctttcttgta caaagtggtg ataacctaga
     3841 gctcgaattt ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg
     3901 ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa
     3961 ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat
     4021 tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc
     4081 gcgcggtgtc atctatgtta ctagatcggg aattctagct tcgacaatta ttttggactc
     4141 tcatgttctc aagctgaaat ccaaaagaaa cgtgatgttg gttcagattt ggaatcactg
     4201 acaatgaagt ttcagtcagc cggtttgtca ccaaggatcc aggcttcatc atgggagccg
     4261 agccgttcaa gtttcaactt cgactatttg gatccgtcct agtggatccc ccatcacaac
     4321 tttgtataga aaagttgaac gagaaacgta aaatgatata aatatcaata tattaaatta
     4381 gattttgcat aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg
     4441 gcggccgcat taggcacccc aggctttaca ctttatgctt ccggctcgta taatgtgtgg
     4501 attttgagtt aggatccggc gagattttca ggagctaagg aagctaaaat ggagaaaaaa
     4561 atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca
     4621 tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt
     4681 ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc
     4741 cgcctgatga atgctcatcc ggaattgcga tcgcatttaa atggccggcc cttaattaag
     4801 gcgcgccgtt taaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4861 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4921 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4981 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5041 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5101 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5161 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc
     5221 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5281 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5341 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5401 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5461 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat
     5521 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5581 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5641 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5701 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5761 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5821 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5881 ccacctgaaa cgggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata
     5941 aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga
     6001 ccaccgatat ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc
     6061 accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag
     6121 gctcccttat acacagccag tctgcaggtc gatcgaccat agtgactgga tatgttgtgt
     6181 tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat
     6241 atcattttac gtttctcgtt caactttatt atacaaagtt gtgatccaca tcttctgatt
     6301 ctattgatct ctcacagttt cagagaggtt tagttgacag gagttcttgt catcctcatg
     6361 taacactgga taaagtgtca aggacacatt tgagagtaga gcaaacaaat agccacatgc
     6421 ctgaaatgaa atcccaacag attacagata ctgaatttga tattcaaaat cacaaagaaa
     6481 gtgctgcttc tctgggaaaa ggaagagaat tgcttccatg ctctgctgca gagtcaataa
     6541 gcactgatgg aggtggtctg atttgttctg aggattcaaa ttggatggca tgttacaaga
     6601 attacccaac tttttaatac aaagttggca ttataagaaa gcattgctta tcaatttgtt
     6661 gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttgccatcca gctgcagctc
     6721 tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga
     6781 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     6841 cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     6901 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc
     6961 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     7021 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     7081 atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc
     7141 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     7201 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     7261 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     7321 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     7381 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     7441 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     7501 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     7561 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     7621 gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg
     7681 acttgacggg acggcgcaag ctcatgacca aaatccctta acgtgagttt tcgttccact
     7741 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     7801 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     7861 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     7921 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     7981 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     8041 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     8101 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     8161 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     8221 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     8281 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     8341 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     8401 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     8461 accgtattac cgctagccag gaagagtttg tagaaacgca aaaaggccat ccgtcaggat
     8521 ggccttctgc ttagtttgat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc
     8581 gggccgttgc ttcacaacgt tcaaatccgc tcccggcgga tttgtcctac tcaggagagc
     8641 gttcaccgac aaacaacaga taaaacgaaa ggcccagtct tccgactgag cctttcgttt
     8701 tatttgatgc ctggcagttc cctactctcg c
//
