LOCUS       40924_36273        5927 bp DNA     circular SYN 18-DEC-2018
DEFINITION  MISSA donor vector pR-ccdB, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5927)
  AUTHORS   Chen QJ, Xie M, Ma XX, Dong L, Chen J, Wang XC.
  TITLE     MISSA is a highly efficient in vivo DNA assembly method for plant 
            multiple-gene transformation
  JOURNAL   Plant Physiol. 153 (1), 41-51 (2010)
  PUBMED    20200068
REFERENCE   2  (bases 1 to 5927)
  AUTHORS   Chen Q-J., Xie M, Ma X-X., Dong L, Chen J, Wang X-C.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-JAN-2010) State Key Laboratory of Plant Physiology and
            Biochemistry, College of Biological Sciences, China Agricultural 
            University, No. 2 West Yuanmingyuan Road, Haidian, Beijing 100193, 
            China
REFERENCE   3  (bases 1 to 5927)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5927)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant 
            Physiol."; date: "2010"; volume: "153"; issue: "1"; pages: "41-51"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (27-JAN-2010) State Key Laboratory of Plant Physiology and 
            Biochemistry, College of Biological Sciences, China Agricultural 
            University, No. 2 West Yuanmingyuan Road, Haidian, Beijing 100193, 
            China"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(1..34)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      100..186
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      278..305
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     primer_bind     373..389
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        397..415
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     protein_bind    complement(430..554)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             714..1016
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRIVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     rep_origin      1169..1757
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(1925..1968)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      2100..2127
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     protein_bind    2210..2334
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2351..2369)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2381..2397)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2471..3889)
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        complement(3890..4335)
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             complement(4473..5129)
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(5182..5212)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     rep_origin      5239..5592
                     /label=oriR6Kgamma
                     /note="oriR6Kgamma"
     oriT            5734..5843
                     /label=oriT
                     /note="incP origin of transfer"
ORIGIN
        1 ataacttcgt atagcataca ttatacgaag ttatgtcgac tctagcatgg atctcgggga
       61 cgtctaacta ctaagcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
      121 tcggaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
      181 acaaatccgc cgggagcgga tttgaacgtt gtgaagcaac ggcccggagg gtggcgggca
      241 ggacgcccgc cataaactgc caggcatcaa actaagcaga aggccatcct gacggatggc
      301 ctttttgcgt ttctacaaac tcttcctgtt agttagttac ttaagcgcca gggttttccc
      361 agtcacgacg ttgtaaaacg acggccagtg catacgattt aggtgacact atagaagggc
      421 ccaatcgacc atagtgactg gatatgttgt gttttacagt attatgtagt ctgtttttta
      481 tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg ttcagctttc
      541 ttgtacaaag tggttcgata attccttaat taacgacttt aggtccaagc ttccctcatg
      601 agcttactaa aagccagata acagtatgcg tatttgcgcg ctgatttttg cggtataaga
      661 atatatactg atatgtatac ccgaagtatg tcaaaaagag gtgtgcttct agaatgcagt
      721 ttaaggttta cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg
      781 atattattga cacgcccggg cgacggatag tgatccccct ggccagtgca cgtctgctgt
      841 cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca
      901 tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc
      961 tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatatagt
     1021 gctgatatcg acccaagtac cgccacctaa caattcgttc aagccgagat cggcttcccg
     1081 gccgcggagt tgttcggtaa attgtcacaa cgccgcccgc cccgttccac tgagcgtcag
     1141 acccggtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     1201 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     1261 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     1321 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     1381 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     1441 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     1501 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     1561 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     1621 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     1681 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     1741 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     1801 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     1861 ccgctagcat ggatctcggg gacgtctaac tactaagcga gagtagggaa ctgccaggca
     1921 tcaaataaaa cgaaaggctc agtcggaaga ctgggccttt cgttttatct gttgtttgtc
     1981 ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgtgaagca
     2041 acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaactaagca
     2101 gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttcctg ttagttagtt
     2161 acttaaggct agcggaattc gacctaaagt cggcgcgcca agctatcaaa caagtttgta
     2221 caaaaaagct gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt
     2281 gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc
     2341 ggctgcaggc cctatagtga gtcgtattac cgtaatcatg gtcatagctg tttcctgtgt
     2401 gaaattgtta tccgctctca tggcggccgc caatgccaat aggatatcgg cattttcttt
     2461 tgcgttttta tttgttaact gttaattgtc cttgttcaag gatgctgtct ttgacaacag
     2521 atgttttctt gcctttgatg ttcagcagga agctaggcgc aaacgttgat tgtttgtctg
     2581 cgtagaatcc tctgtttgtc atatagcttg taatcacgac attgtttcct ttcgcttgag
     2641 gtacagcgaa gtgtgagtaa gtaaaggtta catcgttagg atcaagatcc atttttaaca
     2701 caaggccagt tttgttcagc ggcttgtatg ggccagttaa agaattagaa acataaccaa
     2761 gcatgtaaat atcgttagac gtaatgccgt caatcgtcat ttttgatccg cgcgagtcag
     2821 tgaacaagta ccatttgccg ttcattttaa agacgttcgc gcgttcaatt tcatctgtta
     2881 ctgtgttaga tgcaatcagc ggtttcatca cttttttcag tgtgtaatca tcgtttagct
     2941 caatcatacc gagagcgccg tttgctaact cagccgtgcg ttttttatcg ctttgcagaa
     3001 gtttttgact ttcttgacgg aagaatgatg tgcttttgcc atagtatgct ttgttaaata
     3061 aagattcttc gccttggtag ccatcttcag ttccagtgtt tgcttcaaat actaagtatt
     3121 tgtggccttt atcttctacg tagtgaggat ctctcagcgt atggttgtcg cctgagctgt
     3181 agttgccttc atcgatgaac tgctgtacat tttgatacgt ttttccgtca ccgtcaaaga
     3241 ttgatttata atcctctaca ccgttgatgt tcaaagagct gtctgatgct gatacgttaa
     3301 cttgtgcagt tgtcagtgtt tgtttgccgt aatgtttacc ggagaaatca gtgtagaata
     3361 aacggatttt tccgtcagat gtaaatgtgg ctgaacctga ccattcttgt gtttggtctt
     3421 ttaggataga atcatttgca tcgaatttgt cgctgtcttt aaagacgcgg ccagcgtttt
     3481 tccagctgtc aatagaagtt tcgccgactt tttgatagaa catgtaaatc gatgtgtcat
     3541 ccgcattttt aggatctccg gctaatgcaa agacgatgtg gtagccgtga tagtttgcga
     3601 cagtgccgtc agcgttttgt aatggccagc tgtcccaaac gtccaggcct tttgcagaag
     3661 agatattttt aattgtggac gaatcgaact caggaacttg atatttttca tttttttgct
     3721 gttcagggat ttgcagcata tcatggcgtg taatatggga aatgccgtat gtttccttat
     3781 atggcttttg gttcgtttct ttcgcaaacg cttgagttgc gcctcctgcc agcagtgcgg
     3841 tagtaaaggt taatactgtt gcttgttttg caaacttttt gatgttcatc gttcatgtct
     3901 ccttttttat gtactgtgtt agcggtctgc ttcttccagc cctcctgttt gaagatggca
     3961 agttagttac gcacaataaa aaaagaccta aaatatgtaa ggggtgacgc caaagtatac
     4021 actttgccct ttacacattt taggtcttgc ctgctttatc agtaacaaac ccgcgcgatt
     4081 tacttttcga cctcattcta ttagactctc gtttggattg caactggtct attttcctct
     4141 tttgtttgat agaaaatcat aaaaggattt gcagactacg ggcctaaaga actaaaaaat
     4201 ctatctgttt cttttcattc tctgtatttt ttatagtttc tgttgcatgg gcataaagtt
     4261 gcctttttaa tcacaattca gaaaatatca taatatctca tttcactaaa taatagtgaa
     4321 cggcaggtat atgtggtcga gcctgatagt ttggctgtga gcaattatgt gcttagtgca
     4381 tctaatcgct tgagttaacg ccggcgaagc ggcgtcggct tgaacgaatt tctagctaga
     4441 ggatcgcacc aataactgcc ttaaaaaaat tacgccccgc cctgccactc atcgcagtac
     4501 tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc
     4561 tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc cattgtgaaa
     4621 acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc
     4681 cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg
     4741 ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg
     4801 tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa
     4861 gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg gaactccgga
     4921 tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt
     4981 ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat
     5041 tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg
     5101 gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgac
     5161 gatcctaact caaaatccac acattatacg agccggaagc ataaagtgta aagcctgggg
     5221 gtgcctaatg cgtctagctg cgttacatcc ctggcttgtt gtccacaacc gttaaacctt
     5281 aacagcttta aaagccttat atattctttt ttttcttata aaacttaaaa ccttagaggc
     5341 tatttaagtt gctgatttat attaatttta ttgttcaaac atgagagctt agtacgtgaa
     5401 acatgagagc ttagtacgtt agccatgaga gcttagtacg ttagccatga gggtttagtt
     5461 cgttaaacat gagagcttag tacgttaaac atgagagctt agtacgtgaa acatgagagc
     5521 ttagtacgta ctatcaacag gttgaactgc tgatcttcag atcctctacg ccggacgcat
     5581 cgtggccgga tcaattccgt tttccgctgc ataaccctgc ttcggggtca ttatagcgat
     5641 tttttcggta tatccatcct ttttcgcacg atatacagga ttttgccaaa gggttcgtgt
     5701 agactttcct tggtgtatcc aacggcgtca gccgggcagg ataggtgaag taggcccacc
     5761 cgcgagcggg tgttccttct tcactgtccc ttattcgcac ctggcggtgc tcaacgggaa
     5821 tcctgctctg cgaggctggc cggctaccgc cggcgtaaca gatgagggca agcggatggc
     5881 tgatgaaacc aagccaacca ggaagggcag cccacctatc atcatgg
//
