LOCUS       40924_36618        8140 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-15 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8140)
  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  JOURNAL   Unpublished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 8140)
  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 8140)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8140)
  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            "Unpublished"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..8140
                     /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             1411..1500
                     /codon_start=1
                     /label=3xHA
                     /note="three tandem HA epitope tags"
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    1522..1646
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1683..1713
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1767..2423
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2768..3070
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3114..3238)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      3264..3516
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    3725..3849
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3874..3904
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4176..4216
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4308..5165)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5166..5270)
                     /label=AmpR promoter
     protein_bind    complement(5567..5690)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6016..6111)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6235..7041
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7187..7775
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(7910..7937)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8029..8115)
                     /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 gcgggttaat taacatcttt tacccatacg atgttcctga ctatgcgggc
     1441 tatccctatg acgtcccgga ctatgcagga tcctatccat atgacgttcc agattacgct
     1501 gctcagtgca gcgctgttat cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat
     1561 gatataaata tcaatatatt aaattagatt ttgcataaaa aacagactac ataatactgt
     1621 aaaacacaac atatccagtc actatggcgg cccctcgagg ggccgcatta ggcaccccag
     1681 gctttacact ttatgcttcc ggctcgtata atgtgtggat tttgagttag gatccggcga
     1741 gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg
     1801 atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta
     1861 cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata
     1921 agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg
     1981 aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt
     2041 acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg
     2101 atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg
     2161 cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga
     2221 gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca
     2281 ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc
     2341 atcatgccgt ctgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact
     2401 gcgatgagtg gcagggcggg gcgtaaacgc gtggatccgg cttactaaaa gccagataac
     2461 agtatgcgta tttgcgcgct gatttttgcg gtataagaat atatactgat atgtataccc
     2521 gaagtatgtc aaaaagaggt gtgctatgaa gcagcgtatt acagtgacag ttgacagcga
     2581 cagctatcag ttgctcaagg catatatgat gtcaatatct ccggtctggt aagcacaacc
     2641 atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga aagcggaaaa tcaggaaggg
     2701 atggctgagg tcgcccggtt tattgaaatg aacggctctt ttgctgacga gaacagggac
     2761 tggtgaaatg cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt
     2821 ggatgtacag agtgatatta ttgacacgcc cgggcgacgg atggtgatcc ccctggccag
     2881 tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga
     2941 tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtctccg ttatcgggga
     3001 agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt
     3061 ctggggaata taaatgtcag gctcccttat acacagccag tctgcaggtc gaccatagtg
     3121 actggatatg ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta
     3181 atatattgat atttatatca ttttacgttt ctcgttcagc tttcttgtac aaagtggtga
     3241 taacctagag ctcgaatttc cccgatcgtt caaacatttg gcaataaagt ttcttaagat
     3301 tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc
     3361 atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag
     3421 tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata
     3481 aattatcgcg cgcggtgtca tctatgttac tagatcggga attctagctt cgacaattat
     3541 tttggactct catgttctca agctgaaatc caaaagaaac gtgatgttgg ttcagatttg
     3601 gaatcactga caatgaagtt tcagtcagcc ggtttgtcac caaggatcca ggcttcatca
     3661 tgggagccga gccgttcaag tttcaacttc gactatttgg atccgtccta gtggatcccc
     3721 catcacaact ttgtatagaa aagttgaacg agaaacgtaa aatgatataa atatcaatat
     3781 attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca
     3841 gtcactatgg cggccgcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     3901 aatgtgtgga ttttgagtta ggatccggcg agattttcag gagctaagga agctaaaatg
     3961 gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat
     4021 tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt
     4081 acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac
     4141 attcttgccc gcctgatgaa tgctcatccg gaattgcgat cgcatttaaa tggccggccc
     4201 ttaattaagg cgcgccgttt aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4261 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4321 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4381 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4441 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4501 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4561 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4621 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4681 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4741 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4801 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4861 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4921 acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4981 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5041 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5101 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5161 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5221 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5281 cgaaaagtgc cacctgaaac gggcgacgga tggtgatccc cctggccagt gcacgtctgc
     5341 tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc
     5401 gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg
     5461 atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat
     5521 aaatgtcagg ctcccttata cacagccagt ctgcaggtcg atcgaccata gtgactggat
     5581 atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt
     5641 gatatttata tcattttacg tttctcgttc aactttatta tacaaagttg tgatccacat
     5701 cttctgattc tattgatctc tcacagtttc agagaggttt agttgacagg agttcttgtc
     5761 atcctcatgt aacactggat aaagtgtcaa ggacacattt gagagtagag caaacaaata
     5821 gccacatgcc tgaaatgaaa tcccaacaga ttacagatac tgaatttgat attcaaaatc
     5881 acaaagaaag tgctgcttct ctgggaaaag gaagagaatt gcttccatgc tctgctgcag
     5941 agtcaataag cactgatgga ggtggtctga tttgttctga ggattcaaat tggatggcat
     6001 gttacaagaa ttacccaact ttttaataca aagttggcat tataagaaag cattgcttat
     6061 caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt tgccatccag
     6121 ctgcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata
     6181 tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     6241 catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat
     6301 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
     6361 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     6421 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     6481 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa
     6541 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     6601 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     6661 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     6721 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     6781 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     6841 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     6901 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     6961 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     7021 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc
     7081 attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagtttt
     7141 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     7201 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     7261 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     7321 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     7381 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     7441 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     7501 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     7561 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     7621 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7681 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7741 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7801 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     7861 ctgtggataa ccgtattacc gctagccagg aagagtttgt agaaacgcaa aaaggccatc
     7921 cgtcaggatg gccttctgct tagtttgatg cctggcagtt tatggcgggc gtcctgcccg
     7981 ccaccctccg ggccgttgct tcacaacgtt caaatccgct cccggcggat ttgtcctact
     8041 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt ccgactgagc
     8101 ctttcgtttt atttgatgcc tggcagttcc ctactctcgc
//
