LOCUS       40924_36613        8138 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-14 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8138)
  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  JOURNAL   Unpublished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 8138)
  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 8138)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8138)
  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            "Unpublished"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..8138
                     /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             3139..3228
                     /codon_start=1
                     /label=3xHA
                     /note="three tandem HA epitope tags"
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      3262..3514
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    3723..3847
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3872..3902
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4174..4214
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4306..5163)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5164..5268)
                     /label=AmpR promoter
     protein_bind    complement(5565..5688)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6014..6109)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6233..7039
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7185..7773
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(7908..7935)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8027..8113)
                     /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 gggttaatta acatctttta cccatacgat gttcctgact atgcgggcta tccctatgac
     3181 gtcccggact atgcaggatc ctatccatat gacgttccag attacgctgc tcagtgcagc
     3241 gcttagagct cgaatttccc cgatcgttca aacatttggc aataaagttt cttaagattg
     3301 aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta cgttaagcat
     3361 gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat gattagagtc
     3421 ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa
     3481 ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat tctagcttcg acaattattt
     3541 tggactctca tgttctcaag ctgaaatcca aaagaaacgt gatgttggtt cagatttgga
     3601 atcactgaca atgaagtttc agtcagccgg tttgtcacca aggatccagg cttcatcatg
     3661 ggagccgagc cgttcaagtt tcaacttcga ctatttggat ccgtcctagt ggatccccca
     3721 tcacaacttt gtatagaaaa gttgaacgag aaacgtaaaa tgatataaat atcaatatat
     3781 taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt
     3841 cactatggcg gccgcattag gcaccccagg ctttacactt tatgcttccg gctcgtataa
     3901 tgtgtggatt ttgagttagg atccggcgag attttcagga gctaaggaag ctaaaatgga
     3961 gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt
     4021 tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac
     4081 ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat
     4141 tcttgcccgc ctgatgaatg ctcatccgga attgcgatcg catttaaatg gccggccctt
     4201 aattaaggcg cgccgtttaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4261 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4321 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4381 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4441 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4501 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4561 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc
     4621 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4681 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4741 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4801 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4861 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac
     4921 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4981 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5041 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5101 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5161 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5221 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5281 aaaagtgcca cctgaaacgg gcgacggatg gtgatccccc tggccagtgc acgtctgctg
     5341 tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc
     5401 atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga agtggctgat
     5461 ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa
     5521 atgtcaggct cccttataca cagccagtct gcaggtcgat cgaccatagt gactggatat
     5581 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga
     5641 tatttatatc attttacgtt tctcgttcaa ctttattata caaagttgtg atccacatct
     5701 tctgattcta ttgatctctc acagtttcag agaggtttag ttgacaggag ttcttgtcat
     5761 cctcatgtaa cactggataa agtgtcaagg acacatttga gagtagagca aacaaatagc
     5821 cacatgcctg aaatgaaatc ccaacagatt acagatactg aatttgatat tcaaaatcac
     5881 aaagaaagtg ctgcttctct gggaaaagga agagaattgc ttccatgctc tgctgcagag
     5941 tcaataagca ctgatggagg tggtctgatt tgttctgagg attcaaattg gatggcatgt
     6001 tacaagaatt acccaacttt ttaatacaaa gttggcatta taagaaagca ttgcttatca
     6061 atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct
     6121 gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
     6181 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     6241 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     6301 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     6361 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     6421 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     6481 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac
     6541 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     6601 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     6661 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     6721 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     6781 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     6841 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     6901 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     6961 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     7021 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat
     7081 tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg tgagttttcg
     7141 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7201 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7261 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7321 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7381 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7441 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7501 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7561 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7621 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7681 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7741 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7801 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     7861 gtggataacc gtattaccgc tagccaggaa gagtttgtag aaacgcaaaa aggccatccg
     7921 tcaggatggc cttctgctta gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc
     7981 accctccggg ccgttgcttc acaacgttca aatccgctcc cggcggattt gtcctactca
     8041 ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtcttcc gactgagcct
     8101 ttcgttttat ttgatgcctg gcagttccct actctcgc
//
