LOCUS       40924_36673        8735 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-44 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8735)
  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  JOURNAL   Unpublished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 8735)
  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 8735)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8735)
  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            "Unpublished"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..8735
                     /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             3121..3837
                     /codon_start=1
                     /label=ECFP
                     /note="enhanced CFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      3859..4111
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4320..4444
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4469..4499
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4771..4811
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4903..5760)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5761..5865)
                     /label=AmpR promoter
     protein_bind    complement(6162..6285)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6611..6706)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6830..7636
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7782..8370
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8505..8532)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8624..8710)
                     /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 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     3181 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     3241 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     3301 ctcgtgacca ccctgacctg gggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     3361 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     3421 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     3481 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     3541 aagctggagt acaactacat cagccacaac gtctatatca ccgccgacaa gcagaagaac
     3601 ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     3661 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     3721 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     3781 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggct
     3841 tagagctcga atttccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat
     3901 cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta
     3961 ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg
     4021 caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta
     4081 tcgcgcgcgg tgtcatctat gttactagat cgggaattct agcttcgaca attattttgg
     4141 actctcatgt tctcaagctg aaatccaaaa gaaacgtgat gttggttcag atttggaatc
     4201 actgacaatg aagtttcagt cagccggttt gtcaccaagg atccaggctt catcatggga
     4261 gccgagccgt tcaagtttca acttcgacta tttggatccg tcctagtgga tcccccatca
     4321 caactttgta tagaaaagtt gaacgagaaa cgtaaaatga tataaatatc aatatattaa
     4381 attagatttt gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac
     4441 tatggcggcc gcattaggca ccccaggctt tacactttat gcttccggct cgtataatgt
     4501 gtggattttg agttaggatc cggcgagatt ttcaggagct aaggaagcta aaatggagaa
     4561 aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga
     4621 ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc
     4681 ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct
     4741 tgcccgcctg atgaatgctc atccggaatt gcgatcgcat ttaaatggcc ggcccttaat
     4801 taaggcgcgc cgtttaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     4861 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     4921 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4981 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5041 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5101 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     5161 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctgcagg
     5221 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     5281 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5341 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5401 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5461 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg
     5521 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5581 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     5641 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     5701 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     5761 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     5821 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5881 agtgccacct gaaacgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca
     5941 gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg
     6001 atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc
     6061 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg
     6121 tcaggctccc ttatacacag ccagtctgca ggtcgatcga ccatagtgac tggatatgtt
     6181 gtgttttaca gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat
     6241 ttatatcatt ttacgtttct cgttcaactt tattatacaa agttgtgatc cacatcttct
     6301 gattctattg atctctcaca gtttcagaga ggtttagttg acaggagttc ttgtcatcct
     6361 catgtaacac tggataaagt gtcaaggaca catttgagag tagagcaaac aaatagccac
     6421 atgcctgaaa tgaaatccca acagattaca gatactgaat ttgatattca aaatcacaaa
     6481 gaaagtgctg cttctctggg aaaaggaaga gaattgcttc catgctctgc tgcagagtca
     6541 ataagcactg atggaggtgg tctgatttgt tctgaggatt caaattggat ggcatgttac
     6601 aagaattacc caacttttta atacaaagtt ggcattataa gaaagcattg cttatcaatt
     6661 tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttgcca tccagctgca
     6721 gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc
     6781 atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat
     6841 tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta
     6901 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa
     6961 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac
     7021 agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca
     7081 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc
     7141 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt
     7201 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt
     7261 atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt
     7321 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt
     7381 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
     7441 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     7501 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     7561 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     7621 gctcgatgag tttttctaat cagaattggt taattggttg taacactggc agagcattac
     7681 gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga gttttcgttc
     7741 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     7801 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     7861 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     7921 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7981 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     8041 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     8101 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     8161 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     8221 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     8281 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     8341 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     8401 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     8461 gataaccgta ttaccgctag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca
     8521 ggatggcctt ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc
     8581 ctccgggccg ttgcttcaca acgttcaaat ccgctcccgg cggatttgtc ctactcagga
     8641 gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtcttccgac tgagcctttc
     8701 gttttatttg atgcctggca gttccctact ctcgc
//
