LOCUS       40924_36703        8732 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-60 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8732)
  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  JOURNAL   Unpublished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 8732)
  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 8732)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8732)
  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            "Unpublished"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..8732
                     /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
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(2986..3110)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3121..3831
                     /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"
     terminator      3856..4108
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4317..4441
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4466..4496
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4768..4808
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4900..5757)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5758..5862)
                     /label=AmpR promoter
     protein_bind    complement(6159..6282)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6608..6703)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6827..7633
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7779..8367
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8502..8529)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8621..8707)
                     /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 aggatccggc 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 gtctgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag
     2281 tggcagggcg gggcgtaaag atctggatcc ggcttactaa aagccagata acagtatgcg
     2341 tatttgcgcg ctgatttttg cggtataaga atatatactg atatgtatac ccgaagtatg
     2401 tcaaaaagag gtgtgctatg aagcagcgta ttacagtgac agttgacagc gacagctatc
     2461 agttgctcaa ggcatatatg atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa
     2521 tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag ggatggctga
     2581 ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac gagaacaggg actggtgaaa
     2641 tgcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac
     2701 agagtgatat tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc
     2761 tgctgtcaga taaagtctcc 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 tgataacagc
     3121 atggtgtcta agggcgaaga gctgattaag gagaacatgc acatgaagct gtacatggag
     3181 ggcaccgtga acaaccacca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag
     3241 ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac
     3301 atcctggcta ccagcttcat gtacggcagc agaaccttca tcaaccacac ccagggcatc
     3361 cccgacttct ttaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac
     3421 gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc
     3481 tacaacgtca agatcagagg ggtgaacttc ccatccaacg gccctgtgat gcagaagaaa
     3541 acactcggct gggaggccaa caccgagatg ctgtaccccg ctgacggcgg cctggaaggc
     3601 agaagcgaca tggccctgaa gctcgtgggc gggggccacc tgatctgcaa cttcaagacc
     3661 acatacagat ccaagaaacc cgctaagaac ctcaagatgc ccggcgtcta ctatgtggac
     3721 cacagactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca gcacgaggtg
     3781 gctgtggcca gatactgcga cctccctagc aaactggggc acaaacttaa ttgagcttag
     3841 agctcgaatt tccccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct
     3901 gttgccggtc ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata
     3961 attaacatgt aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa
     4021 ttatacattt aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg
     4081 cgcgcggtgt catctatgtt actagatcgg gaattctagc ttcgacaatt attttggact
     4141 ctcatgttct caagctgaaa tccaaaagaa acgtgatgtt ggttcagatt tggaatcact
     4201 gacaatgaag tttcagtcag ccggtttgtc accaaggatc caggcttcat catgggagcc
     4261 gagccgttca agtttcaact tcgactattt ggatccgtcc tagtggatcc cccatcacaa
     4321 ctttgtatag aaaagttgaa cgagaaacgt aaaatgatat aaatatcaat atattaaatt
     4381 agattttgca taaaaaacag actacataat actgtaaaac acaacatatc cagtcactat
     4441 ggcggccgca ttaggcaccc caggctttac actttatgct tccggctcgt ataatgtgtg
     4501 gattttgagt taggatccgg cgagattttc aggagctaag gaagctaaaa tggagaaaaa
     4561 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc
     4621 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt
     4681 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc
     4741 ccgcctgatg aatgctcatc cggaattgcg atcgcattta aatggccggc ccttaattaa
     4801 ggcgcgccgt ttaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4861 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4921 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4981 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5041 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5101 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5161 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat
     5221 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5281 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5341 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5401 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5461 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga
     5521 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5581 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5641 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5701 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5761 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5821 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5881 gccacctgaa acgggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat
     5941 aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg
     6001 accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc
     6061 caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaatgtca
     6121 ggctccctta tacacagcca gtctgcaggt cgatcgacca tagtgactgg atatgttgtg
     6181 ttttacagta ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta
     6241 tatcatttta cgtttctcgt tcaactttat tatacaaagt tgtgatccac atcttctgat
     6301 tctattgatc tctcacagtt tcagagaggt ttagttgaca ggagttcttg tcatcctcat
     6361 gtaacactgg ataaagtgtc aaggacacat ttgagagtag agcaaacaaa tagccacatg
     6421 cctgaaatga aatcccaaca gattacagat actgaatttg atattcaaaa tcacaaagaa
     6481 agtgctgctt ctctgggaaa aggaagagaa ttgcttccat gctctgctgc agagtcaata
     6541 agcactgatg gaggtggtct gatttgttct gaggattcaa attggatggc atgttacaag
     6601 aattacccaa ctttttaata caaagttggc attataagaa agcattgctt atcaatttgt
     6661 tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttgccatcc agctgcagct
     6721 ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg
     6781 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     6841 acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     6901 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc
     6961 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     7021 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     7081 tatccgtact cctgatgatg catggttact caccactgcg atccccggaa aaacagcatt
     7141 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     7201 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     7261 tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     7321 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc
     7381 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     7441 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     7501 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     7561 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     7621 cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct
     7681 gacttgacgg gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     7741 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     7801 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     7861 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     7921 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     7981 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     8041 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     8101 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     8161 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     8221 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     8281 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     8341 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     8401 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     8461 aaccgtatta ccgctagcca ggaagagttt gtagaaacgc aaaaaggcca tccgtcagga
     8521 tggccttctg cttagtttga tgcctggcag tttatggcgg gcgtcctgcc cgccaccctc
     8581 cgggccgttg cttcacaacg ttcaaatccg ctcccggcgg atttgtccta ctcaggagag
     8641 cgttcaccga caaacaacag ataaaacgaa aggcccagtc ttccgactga gcctttcgtt
     8701 ttatttgatg cctggcagtt ccctactctc gc
//
