LOCUS       40924_36698        8695 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-55 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8695)
  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  JOURNAL   Unpublished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 8695)
  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 8695)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8695)
  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            "Unpublished"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..8695
                     /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..2067
                     /codon_start=1
                     /label=mRFP1
                     /note="monomeric derivative of DsRed (Campbell et al.,
                     2002)"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     protein_bind    2077..2201
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2238..2268
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             2322..2978
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             3323..3625
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3669..3793)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      3819..4071
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4280..4404
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4429..4459
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4731..4771
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4863..5720)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5721..5825)
                     /label=AmpR promoter
     protein_bind    complement(6122..6245)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6571..6666)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6790..7596
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7742..8330
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8465..8492)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8584..8670)
                     /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 gcatggcctc ctccgaggac gtcatcaagg agttcatgcg cttcaaggtg
     1441 cgcatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     1501 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc
     1561 gcctgggaca tcctgtcccc tcagttccag tacggctcca aggcctacgt gaagcacccc
     1621 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     1681 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     1741 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     1801 cagaagaaga ccatgggctg ggaggcctcc accgagcgga tgtaccccga ggacggcgcc
     1861 ctgaagggcg agatcaagat gaggctgaag ctgaaggacg gcggccacta cgacgccgag
     1921 gtcaagacca cctacatggc caagaagccc gtgcagctgc ccggcgccta caagaccgac
     1981 atcaagctgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc
     2041 gccgagggcc gccactccac cggcgccgct gttatcacaa gtttgtacaa aaaagctgaa
     2101 cgagaaacgt aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag
     2161 actacataat actgtaaaac acaacatatc cagtcactat ggcggcccct cgaggggccg
     2221 cattaggcac cccaggcttt acactttatg cttccggctc gtataatgtg tggattttga
     2281 gttaggatcc ggcgagattt tcaggagcta aggaagctaa aatggagaaa aaaatcactg
     2341 gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag gcatttcagt
     2401 cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc tttttaaaga
     2461 ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt gcccgcctga
     2521 tgaatgctca tccggaattc cgtatggcaa tgaaagacgg tgagctggtg atatgggata
     2581 gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga
     2641 gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt
     2701 acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt ttcgtctcag
     2761 ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg gacaacttct
     2821 tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc
     2881 tggcgattca ggttcatcat gccgtctgtg atggcttcca tgtcggcaga atgcttaatg
     2941 aattacaaca gtactgcgat gagtggcagg gcggggcgta aacgcgtgga tccggcttac
     3001 taaaagccag ataacagtat gcgtatttgc gcgctgattt ttgcggtata agaatatata
     3061 ctgatatgta tacccgaagt atgtcaaaaa gaggtgtgct atgaagcagc gtattacagt
     3121 gacagttgac agcgacagct atcagttgct caaggcatat atgatgtcaa tatctccggt
     3181 ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg ctggaaagcg
     3241 gaaaatcagg aagggatggc tgaggtcgcc cggtttattg aaatgaacgg ctcttttgct
     3301 gacgagaaca gggactggtg aaatgcagtt taaggtttac acctataaaa gagagagccg
     3361 ttatcgtctg tttgtggatg tacagagtga tattattgac acgcccgggc gacggatggt
     3421 gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac tttacccggt
     3481 ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt
     3541 ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc
     3601 cattaacctg atgttctggg gaatataaat gtcaggctcc cttatacaca gccagtctgc
     3661 aggtcgacca tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat
     3721 gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt tcagctttct
     3781 tgtacaaagt ggtgataacc tagagctcga atttccccga tcgttcaaac atttggcaat
     3841 aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt
     3901 tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg
     3961 tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc
     4021 gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattct
     4081 agcttcgaca attattttgg actctcatgt tctcaagctg aaatccaaaa gaaacgtgat
     4141 gttggttcag atttggaatc actgacaatg aagtttcagt cagccggttt gtcaccaagg
     4201 atccaggctt catcatggga gccgagccgt tcaagtttca acttcgacta tttggatccg
     4261 tcctagtgga tcccccatca caactttgta tagaaaagtt gaacgagaaa cgtaaaatga
     4321 tataaatatc aatatattaa attagatttt gcataaaaaa cagactacat aatactgtaa
     4381 aacacaacat atccagtcac tatggcggcc gcattaggca ccccaggctt tacactttat
     4441 gcttccggct cgtataatgt gtggattttg agttaggatc cggcgagatt ttcaggagct
     4501 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg
     4561 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc
     4621 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat
     4681 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt gcgatcgcat
     4741 ttaaatggcc ggcccttaat taaggcgcgc cgtttaagga tcttcaccta gatcctttta
     4801 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4861 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4921 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4981 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5041 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5101 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5161 gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5221 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5281 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5341 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5401 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5461 tcttgcccgg cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5521 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5581 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5641 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5701 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5761 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5821 ccgcgcacat ttccccgaaa agtgccacct gaaacgggcg acggatggtg atccccctgg
     5881 ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg
     5941 gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg
     6001 gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga
     6061 tgttctgggg aatataaatg tcaggctccc ttatacacag ccagtctgca ggtcgatcga
     6121 ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat
     6181 ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcaactt tattatacaa
     6241 agttgtgatc cacatcttct gattctattg atctctcaca gtttcagaga ggtttagttg
     6301 acaggagttc ttgtcatcct catgtaacac tggataaagt gtcaaggaca catttgagag
     6361 tagagcaaac aaatagccac atgcctgaaa tgaaatccca acagattaca gatactgaat
     6421 ttgatattca aaatcacaaa gaaagtgctg cttctctggg aaaaggaaga gaattgcttc
     6481 catgctctgc tgcagagtca ataagcactg atggaggtgg tctgatttgt tctgaggatt
     6541 caaattggat ggcatgttac aagaattacc caacttttta atacaaagtt ggcattataa
     6601 gaaagcattg cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa aataaaatca
     6661 ttatttgcca tccagctgca gctctggccc gtgtctcaaa atctctgatg ttacattgca
     6721 caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     6781 aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg
     6841 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     6901 atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     6961 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     7021 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     7081 gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     7141 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     7201 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     7261 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     7321 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     7381 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     7441 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     7501 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     7561 aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg
     7621 taacactggc agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc
     7681 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     7741 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     7801 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     7861 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     7921 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     7981 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     8041 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     8101 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     8161 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     8221 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     8281 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     8341 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     8401 cgttatcccc tgattctgtg gataaccgta ttaccgctag ccaggaagag tttgtagaaa
     8461 cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt tgatgcctgg cagtttatgg
     8521 cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca acgttcaaat ccgctcccgg
     8581 cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca
     8641 gtcttccgac tgagcctttc gttttatttg atgcctggca gttccctact ctcgc
//
