LOCUS       40924_36668        8737 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-42 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8737)
  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  JOURNAL   Unpublished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 8737)
  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 8737)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8737)
  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            "Unpublished"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..8737
                     /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..2109
                     /codon_start=1
                     /label=EYFP
                     /note="enhanced YFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     protein_bind    2119..2243
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2280..2310
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             2364..3020
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             3365..3667
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3711..3835)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      3861..4113
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4322..4446
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4471..4501
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4773..4813
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4905..5762)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5763..5867)
                     /label=AmpR promoter
     protein_bind    complement(6164..6287)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6613..6708)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6832..7638
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7784..8372
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8507..8534)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8626..8712)
                     /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 gcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1441 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1501 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1561 ccctggccca ccctcgtgac caccttcggc tacggcctgc agtgcttcgc ccgctacccc
     1621 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1681 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1741 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1801 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1861 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1921 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1981 cccgacaacc actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc
     2041 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     2101 ctgtacaagg ctgttatcac aagtttgtac aaaaaagctg aacgagaaac gtaaaatgat
     2161 ataaatatca atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa
     2221 acacaacata tccagtcact atggcggccc ctcgaggggc cgcattaggc accccaggct
     2281 ttacacttta tgcttccggc tcgtataatg tgtggatttt gagttaggat ccggcgagat
     2341 tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata
     2401 tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct
     2461 ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc
     2521 acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat
     2581 tccgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca
     2641 ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt
     2701 tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct
     2761 atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt
     2821 tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca
     2881 tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc
     2941 atgccgtctg tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg
     3001 atgagtggca gggcggggcg taaacgcgtg gatccggctt actaaaagcc agataacagt
     3061 atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg tatacccgaa
     3121 gtatgtcaaa aagaggtgtg ctatgaagca gcgtattaca gtgacagttg acagcgacag
     3181 ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtaag cacaaccatg
     3241 cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca ggaagggatg
     3301 gctgaggtcg cccggtttat tgaaatgaac ggctcttttg ctgacgagaa cagggactgg
     3361 tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga
     3421 tgtacagagt gatattattg acacgcccgg gcgacggatg gtgatccccc tggccagtgc
     3481 acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga
     3541 aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga
     3601 agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg
     3661 gggaatataa atgtcaggct cccttataca cagccagtct gcaggtcgac catagtgact
     3721 ggatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata
     3781 tattgatatt tatatcattt tacgtttctc gttcagcttt cttgtacaaa gtggtgataa
     3841 cctagagctc gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga
     3901 atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg
     3961 taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc
     4021 cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat
     4081 tatcgcgcgc ggtgtcatct atgttactag atcgggaatt ctagcttcga caattatttt
     4141 ggactctcat gttctcaagc tgaaatccaa aagaaacgtg atgttggttc agatttggaa
     4201 tcactgacaa tgaagtttca gtcagccggt ttgtcaccaa ggatccaggc ttcatcatgg
     4261 gagccgagcc gttcaagttt caacttcgac tatttggatc cgtcctagtg gatcccccat
     4321 cacaactttg tatagaaaag ttgaacgaga aacgtaaaat gatataaata tcaatatatt
     4381 aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc
     4441 actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat
     4501 gtgtggattt tgagttagga tccggcgaga ttttcaggag ctaaggaagc taaaatggag
     4561 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
     4621 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
     4681 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
     4741 cttgcccgcc tgatgaatgc tcatccggaa ttgcgatcgc atttaaatgg ccggccctta
     4801 attaaggcgc gccgtttaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4861 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4921 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4981 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5041 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5101 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5161 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca
     5221 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5281 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5341 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5401 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5461 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca
     5521 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5581 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5641 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5701 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5761 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5821 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5881 aaagtgccac ctgaaacggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt
     5941 cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca
     6001 tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc
     6061 tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataaa
     6121 tgtcaggctc ccttatacac agccagtctg caggtcgatc gaccatagtg actggatatg
     6181 ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta atatattgat
     6241 atttatatca ttttacgttt ctcgttcaac tttattatac aaagttgtga tccacatctt
     6301 ctgattctat tgatctctca cagtttcaga gaggtttagt tgacaggagt tcttgtcatc
     6361 ctcatgtaac actggataaa gtgtcaagga cacatttgag agtagagcaa acaaatagcc
     6421 acatgcctga aatgaaatcc caacagatta cagatactga atttgatatt caaaatcaca
     6481 aagaaagtgc tgcttctctg ggaaaaggaa gagaattgct tccatgctct gctgcagagt
     6541 caataagcac tgatggaggt ggtctgattt gttctgagga ttcaaattgg atggcatgtt
     6601 acaagaatta cccaactttt taatacaaag ttggcattat aagaaagcat tgcttatcaa
     6661 tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc catccagctg
     6721 cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca
     6781 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     6841 attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg
     6901 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg
     6961 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     7021 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
     7081 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca
     7141 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
     7201 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
     7261 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
     7321 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt
     7381 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
     7441 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     7501 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     7561 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     7621 atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt
     7681 acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttttcgt
     7741 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     7801 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     7861 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     7921 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     7981 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     8041 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     8101 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     8161 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     8221 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     8281 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     8341 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     8401 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     8461 tggataaccg tattaccgct agccaggaag agtttgtaga aacgcaaaaa ggccatccgt
     8521 caggatggcc ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca
     8581 ccctccgggc cgttgcttca caacgttcaa atccgctccc ggcggatttg tcctactcag
     8641 gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtcttccg actgagcctt
     8701 tcgttttatt tgatgcctgg cagttcccta ctctcgc
//
