LOCUS       40924_36598        8038 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-09 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8038)
  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  JOURNAL   Unpublished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 8038)
  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 8038)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8038)
  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            "Unpublished"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..8038
                     /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..1410
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     protein_bind    1420..1544
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1581..1611
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1665..2321
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2666..2968
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3012..3136)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      3162..3414
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    3623..3747
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3772..3802
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4074..4114
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4206..5063)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5064..5168)
                     /label=AmpR promoter
     protein_bind    complement(5465..5588)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(5914..6009)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6133..6939
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7085..7673
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(7808..7835)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(7927..8013)
                     /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 gccatcatca tcatcatcat gctgttatca caagtttgta caaaaaagct
     1441 gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa
     1501 cagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc cctcgagggg
     1561 ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat gtgtggattt
     1621 tgagttagga tccggcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca
     1681 ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc
     1741 agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa
     1801 agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc
     1861 tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg
     1921 atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct
     1981 ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt
     2041 gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct
     2101 cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact
     2161 tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc
     2221 cgctggcgat tcaggttcat catgccgtct gtgatggctt ccatgtcggc agaatgctta
     2281 atgaattaca acagtactgc gatgagtggc agggcggggc gtaaacgcgt ggatccggct
     2341 tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat
     2401 atactgatat gtatacccga agtatgtcaa aaagaggtgt gctatgaagc agcgtattac
     2461 agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc
     2521 ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa
     2581 gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt
     2641 gctgacgaga acagggactg gtgaaatgca gtttaaggtt tacacctata aaagagagag
     2701 ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat
     2761 ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc
     2821 ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc
     2881 ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa
     2941 cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac acagccagtc
     3001 tgcaggtcga ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt
     3061 tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt
     3121 tcttgtacaa agtggtgata acctagagct cgaatttccc cgatcgttca aacatttggc
     3181 aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc
     3241 tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat
     3301 gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat
     3361 agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat
     3421 tctagcttcg acaattattt tggactctca tgttctcaag ctgaaatcca aaagaaacgt
     3481 gatgttggtt cagatttgga atcactgaca atgaagtttc agtcagccgg tttgtcacca
     3541 aggatccagg cttcatcatg ggagccgagc cgttcaagtt tcaacttcga ctatttggat
     3601 ccgtcctagt ggatccccca tcacaacttt gtatagaaaa gttgaacgag aaacgtaaaa
     3661 tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta cataatactg
     3721 taaaacacaa catatccagt cactatggcg gccgcattag gcaccccagg ctttacactt
     3781 tatgcttccg gctcgtataa tgtgtggatt ttgagttagg atccggcgag attttcagga
     3841 gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa
     3901 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag
     3961 accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt
     4021 tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attgcgatcg
     4081 catttaaatg gccggccctt aattaaggcg cgccgtttaa ggatcttcac ctagatcctt
     4141 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4201 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4261 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4321 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     4381 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4441 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     4501 aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     4561 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     4621 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     4681 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     4741 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     4801 tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg
     4861 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     4921 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     4981 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5041 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5101 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5161 gttccgcgca catttccccg aaaagtgcca cctgaaacgg gcgacggatg gtgatccccc
     5221 tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata
     5281 tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtctccgtta
     5341 tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc
     5401 tgatgttctg gggaatataa atgtcaggct cccttataca cagccagtct gcaggtcgat
     5461 cgaccatagt gactggatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa
     5521 aatctaattt aatatattga tatttatatc attttacgtt tctcgttcaa ctttattata
     5581 caaagttgtg atccacatct tctgattcta ttgatctctc acagtttcag agaggtttag
     5641 ttgacaggag ttcttgtcat cctcatgtaa cactggataa agtgtcaagg acacatttga
     5701 gagtagagca aacaaatagc cacatgcctg aaatgaaatc ccaacagatt acagatactg
     5761 aatttgatat tcaaaatcac aaagaaagtg ctgcttctct gggaaaagga agagaattgc
     5821 ttccatgctc tgctgcagag tcaataagca ctgatggagg tggtctgatt tgttctgagg
     5881 attcaaattg gatggcatgt tacaagaatt acccaacttt ttaatacaaa gttggcatta
     5941 taagaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa
     6001 tcattatttg ccatccagct gcagctctgg cccgtgtctc aaaatctctg atgttacatt
     6061 gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat
     6121 acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt aaattccaac
     6181 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     6241 acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     6301 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     6361 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     6421 actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     6481 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     6541 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     6601 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     6661 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     6721 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     6781 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     6841 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     6901 aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt ggttaattgg
     6961 ttgtaacact ggcagagcat tacgctgact tgacgggacg gcgcaagctc atgaccaaaa
     7021 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7081 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     7141 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     7201 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     7261 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     7321 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     7381 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7441 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7501 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7561 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7621 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7681 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     7741 ctgcgttatc ccctgattct gtggataacc gtattaccgc tagccaggaa gagtttgtag
     7801 aaacgcaaaa aggccatccg tcaggatggc cttctgctta gtttgatgcc tggcagttta
     7861 tggcgggcgt cctgcccgcc accctccggg ccgttgcttc acaacgttca aatccgctcc
     7921 cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc
     7981 ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgc
//
