LOCUS       40924_36603        8042 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-11 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8042)
  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  JOURNAL   Unpublished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 8042)
  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 8042)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8042)
  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            "Unpublished"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..8042
                     /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..3144
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     terminator      3166..3418
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    3627..3751
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3776..3806
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4078..4118
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4210..5067)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5068..5172)
                     /label=AmpR promoter
     protein_bind    complement(5469..5592)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(5918..6013)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6137..6943
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7089..7677
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(7812..7839)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(7931..8017)
                     /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 gactacaagg atgacgatga caaggcttag agctcgaatt tccccgatcg ttcaaacatt
     3181 tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa
     3241 tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg
     3301 agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa
     3361 atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg
     3421 gaattctagc ttcgacaatt attttggact ctcatgttct caagctgaaa tccaaaagaa
     3481 acgtgatgtt ggttcagatt tggaatcact gacaatgaag tttcagtcag ccggtttgtc
     3541 accaaggatc caggcttcat catgggagcc gagccgttca agtttcaact tcgactattt
     3601 ggatccgtcc tagtggatcc cccatcacaa ctttgtatag aaaagttgaa cgagaaacgt
     3661 aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag actacataat
     3721 actgtaaaac acaacatatc cagtcactat ggcggccgca ttaggcaccc caggctttac
     3781 actttatgct tccggctcgt ataatgtgtg gattttgagt taggatccgg cgagattttc
     3841 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc
     3901 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa
     3961 ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa
     4021 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattgcg
     4081 atcgcattta aatggccggc ccttaattaa ggcgcgccgt ttaaggatct tcacctagat
     4141 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4201 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4261 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4321 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4381 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4441 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4501 gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4561 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4621 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4681 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4741 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4801 gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca gaactttaaa
     4861 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4921 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4981 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5041 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5101 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5161 aggggttccg cgcacatttc cccgaaaagt gccacctgaa acgggcgacg gatggtgatc
     5221 cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg
     5281 catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc
     5341 gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt
     5401 aacctgatgt tctggggaat ataaatgtca ggctccctta tacacagcca gtctgcaggt
     5461 cgatcgacca tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat
     5521 gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt tcaactttat
     5581 tatacaaagt tgtgatccac atcttctgat tctattgatc tctcacagtt tcagagaggt
     5641 ttagttgaca ggagttcttg tcatcctcat gtaacactgg ataaagtgtc aaggacacat
     5701 ttgagagtag agcaaacaaa tagccacatg cctgaaatga aatcccaaca gattacagat
     5761 actgaatttg atattcaaaa tcacaaagaa agtgctgctt ctctgggaaa aggaagagaa
     5821 ttgcttccat gctctgctgc agagtcaata agcactgatg gaggtggtct gatttgttct
     5881 gaggattcaa attggatggc atgttacaag aattacccaa ctttttaata caaagttggc
     5941 attataagaa agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat
     6001 aaaatcatta tttgccatcc agctgcagct ctggcccgtg tctcaaaatc tctgatgtta
     6061 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag
     6121 taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc
     6181 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     6241 tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     6301 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     6361 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     6421 caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     6481 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     6541 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     6601 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     6661 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     6721 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     6781 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     6841 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     6901 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa
     6961 ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc
     7021 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     7081 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     7141 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     7201 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     7261 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     7321 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     7381 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     7441 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     7501 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7561 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7621 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7681 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     7741 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgctagcca ggaagagttt
     7801 gtagaaacgc aaaaaggcca tccgtcagga tggccttctg cttagtttga tgcctggcag
     7861 tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg ttcaaatccg
     7921 ctcccggcgg atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa
     7981 aggcccagtc ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc
     8041 gc
//
