LOCUS       40924_36578        8018 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-02 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8018)
  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  JOURNAL   Unpublished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 8018)
  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 8018)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8018)
  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            "Unpublished"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..8018
                     /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"
     terminator      3142..3394
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    3603..3727
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3752..3782
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4054..4094
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4186..5043)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5044..5148)
                     /label=AmpR promoter
     protein_bind    complement(5445..5568)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(5894..5989)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6113..6919
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7065..7653
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(7788..7815)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(7907..7993)
                     /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 ctgcaccgga
     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 gcttagagct cgaatttccc cgatcgttca aacatttggc aataaagttt cttaagattg
     3181 aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta cgttaagcat
     3241 gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat gattagagtc
     3301 ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa
     3361 ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat tctagcttcg acaattattt
     3421 tggactctca tgttctcaag ctgaaatcca aaagaaacgt gatgttggtt cagatttgga
     3481 atcactgaca atgaagtttc agtcagccgg tttgtcacca aggatccagg cttcatcatg
     3541 ggagccgagc cgttcaagtt tcaacttcga ctatttggat ccgtcctagt ggatccccca
     3601 tcacaacttt gtatagaaaa gttgaacgag aaacgtaaaa tgatataaat atcaatatat
     3661 taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt
     3721 cactatggcg gccgcattag gcaccccagg ctttacactt tatgcttccg gctcgtataa
     3781 tgtgtggatt ttgagttagg atccggcgag attttcagga gctaaggaag ctaaaatgga
     3841 gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt
     3901 tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac
     3961 ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat
     4021 tcttgcccgc ctgatgaatg ctcatccgga attgcgatcg catttaaatg gccggccctt
     4081 aattaaggcg cgccgtttaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4141 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4201 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4261 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4321 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4381 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4441 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc
     4501 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4561 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4621 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4681 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4741 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac
     4801 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4861 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4921 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4981 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5041 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5101 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5161 aaaagtgcca cctgaaacgg gcgacggatg gtgatccccc tggccagtgc acgtctgctg
     5221 tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc
     5281 atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga agtggctgat
     5341 ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa
     5401 atgtcaggct cccttataca cagccagtct gcaggtcgat cgaccatagt gactggatat
     5461 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga
     5521 tatttatatc attttacgtt tctcgttcaa ctttattata caaagttgtg atccacatct
     5581 tctgattcta ttgatctctc acagtttcag agaggtttag ttgacaggag ttcttgtcat
     5641 cctcatgtaa cactggataa agtgtcaagg acacatttga gagtagagca aacaaatagc
     5701 cacatgcctg aaatgaaatc ccaacagatt acagatactg aatttgatat tcaaaatcac
     5761 aaagaaagtg ctgcttctct gggaaaagga agagaattgc ttccatgctc tgctgcagag
     5821 tcaataagca ctgatggagg tggtctgatt tgttctgagg attcaaattg gatggcatgt
     5881 tacaagaatt acccaacttt ttaatacaaa gttggcatta taagaaagca ttgcttatca
     5941 atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct
     6001 gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
     6061 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     6121 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     6181 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     6241 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     6301 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     6361 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac
     6421 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     6481 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     6541 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     6601 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     6661 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     6721 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     6781 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     6841 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     6901 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat
     6961 tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg tgagttttcg
     7021 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7081 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7141 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7201 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7261 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7321 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7381 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7441 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7501 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7561 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7621 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7681 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     7741 gtggataacc gtattaccgc tagccaggaa gagtttgtag aaacgcaaaa aggccatccg
     7801 tcaggatggc cttctgctta gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc
     7861 accctccggg ccgttgcttc acaacgttca aatccgctcc cggcggattt gtcctactca
     7921 ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtcttcc gactgagcct
     7981 ttcgttttat ttgatgcctg gcagttccct actctcgc
//
