LOCUS       40924_36688        8734 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-52 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8734)
  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  JOURNAL   Unpublished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 8734)
  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 8734)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8734)
  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            "Unpublished"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..8734
                     /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..2106
                     /codon_start=1
                     /label=GFP
                     /note="Aequorea victoria green fluorescent protein"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFAYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     protein_bind    2116..2240
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2277..2307
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             2361..3017
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             3362..3664
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3708..3832)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      3858..4110
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4319..4443
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4468..4498
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4770..4810
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4902..5759)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5760..5864)
                     /label=AmpR promoter
     protein_bind    complement(6161..6284)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6610..6705)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6829..7635
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7781..8369
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8504..8531)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8623..8709)
                     /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 gcatgagtaa aggagaagaa cttttcactg gagttgtccc aattcttgtt
     1441 gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat
     1501 gcaacatacg gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttcca
     1561 tggccaacac ttgttactac tttcgcttac ggtgtgcagt gcttctcaag atacccagat
     1621 catatgaagc ggcacgactt cttcaagagc gccatgcctg agggatacgt gcaggagagg
     1681 accatcttct tcaaggacga cgggaactac aagacacgtg ctgaagtcaa gtttgaggga
     1741 gacaccctcg tcaacaggat cgagcttaag ggaatcgatt tcaaggagga cggaaacatc
     1801 ctcggccaca agttggaata caacttcaac tcccacaacg tgtacatcat ggcagacaaa
     1861 caaaagaatg gaatcaaagt taacttcaaa attagacaca acattgaaga tggaagcgtt
     1921 caactagcag accattatca acaaaatact ccaattggcg atggccctgt ccttttacca
     1981 gacaaccatt acctgtccac acaatctgcc ctttcgaaag atcccaacga aaagagagac
     2041 cacatggtcc ttcttgagtt tgtaacagct gctgggatta cacatggcat ggatgaacta
     2101 tacaaagctg ttatcacaag tttgtacaaa aaagctgaac gagaaacgta aaatgatata
     2161 aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca
     2221 caacatatcc agtcactatg gcggcccctc gaggggccgc attaggcacc ccaggcttta
     2281 cactttatgc ttccggctcg tataatgtgt ggattttgag ttaggatccg gcgagatttt
     2341 caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat
     2401 cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata
     2461 accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca
     2521 agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattcc
     2581 gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg
     2641 ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc
     2701 ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt
     2761 tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca
     2821 ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg
     2881 gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg
     2941 ccgtctgtga tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg
     3001 agtggcaggg cggggcgtaa acgcgtggat ccggcttact aaaagccaga taacagtatg
     3061 cgtatttgcg cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta
     3121 tgtcaaaaag aggtgtgcta tgaagcagcg tattacagtg acagttgaca gcgacagcta
     3181 tcagttgctc aaggcatata tgatgtcaat atctccggtc tggtaagcac aaccatgcag
     3241 aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga agggatggct
     3301 gaggtcgccc ggtttattga aatgaacggc tcttttgctg acgagaacag ggactggtga
     3361 aatgcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt
     3421 acagagtgat attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg
     3481 tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag
     3541 ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt
     3601 ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg
     3661 aatataaatg tcaggctccc ttatacacag ccagtctgca ggtcgaccat agtgactgga
     3721 tatgttgtgt tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat
     3781 tgatatttat atcattttac gtttctcgtt cagctttctt gtacaaagtg gtgataacct
     3841 agagctcgaa tttccccgat cgttcaaaca tttggcaata aagtttctta agattgaatc
     3901 ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa
     3961 taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc
     4021 aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat
     4081 cgcgcgcggt gtcatctatg ttactagatc gggaattcta gcttcgacaa ttattttgga
     4141 ctctcatgtt ctcaagctga aatccaaaag aaacgtgatg ttggttcaga tttggaatca
     4201 ctgacaatga agtttcagtc agccggtttg tcaccaagga tccaggcttc atcatgggag
     4261 ccgagccgtt caagtttcaa cttcgactat ttggatccgt cctagtggat cccccatcac
     4321 aactttgtat agaaaagttg aacgagaaac gtaaaatgat ataaatatca atatattaaa
     4381 ttagattttg cataaaaaac agactacata atactgtaaa acacaacata tccagtcact
     4441 atggcggccg cattaggcac cccaggcttt acactttatg cttccggctc gtataatgtg
     4501 tggattttga gttaggatcc ggcgagattt tcaggagcta aggaagctaa aatggagaaa
     4561 aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag
     4621 gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc
     4681 tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt
     4741 gcccgcctga tgaatgctca tccggaattg cgatcgcatt taaatggccg gcccttaatt
     4801 aaggcgcgcc gtttaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4861 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4921 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4981 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5041 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5101 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5161 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc
     5221 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5281 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5341 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5401 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5461 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg
     5521 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5581 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5641 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5701 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5761 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5821 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5881 gtgccacctg aaacgggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag
     5941 ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga
     6001 tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca
     6061 gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt
     6121 caggctccct tatacacagc cagtctgcag gtcgatcgac catagtgact ggatatgttg
     6181 tgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata tattgatatt
     6241 tatatcattt tacgtttctc gttcaacttt attatacaaa gttgtgatcc acatcttctg
     6301 attctattga tctctcacag tttcagagag gtttagttga caggagttct tgtcatcctc
     6361 atgtaacact ggataaagtg tcaaggacac atttgagagt agagcaaaca aatagccaca
     6421 tgcctgaaat gaaatcccaa cagattacag atactgaatt tgatattcaa aatcacaaag
     6481 aaagtgctgc ttctctggga aaaggaagag aattgcttcc atgctctgct gcagagtcaa
     6541 taagcactga tggaggtggt ctgatttgtt ctgaggattc aaattggatg gcatgttaca
     6601 agaattaccc aactttttaa tacaaagttg gcattataag aaagcattgc ttatcaattt
     6661 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgcag
     6721 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     6781 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     6841 caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     6901 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag
     6961 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     7021 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     7081 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca
     7141 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     7201 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     7261 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     7321 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     7381 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     7441 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     7501 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     7561 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     7621 ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg
     7681 ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     7741 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     7801 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     7861 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     7921 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     7981 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     8041 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     8101 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     8161 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     8221 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     8281 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     8341 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     8401 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     8461 ataaccgtat taccgctagc caggaagagt ttgtagaaac gcaaaaaggc catccgtcag
     8521 gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg cccgccaccc
     8581 tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag
     8641 agcgttcacc gacaaacaac agataaaacg aaaggcccag tcttccgact gagcctttcg
     8701 ttttatttga tgcctggcag ttccctactc tcgc
//
