LOCUS       40924_27169        5857 bp DNA     circular SYN 18-DEC-2018
DEFINITION  MISSA donor vector pL-ccdB, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5857)
  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 5857)
  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 5857)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5857)
  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..5857
                     /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    423..522
                     /label=attL1
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             673..975
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRIVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     rep_origin      1128..1716
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(1884..1927)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      2059..2086
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     protein_bind    complement(2165..2264)
                     /label=attL2
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2281..2299)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2311..2327)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2401..3819)
                     /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(3820..4265)
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             complement(4403..5059)
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(5112..5142)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     rep_origin      5169..5522
                     /label=oriR6Kgamma
                     /note="oriR6Kgamma"
     oriT            5664..5773
                     /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 cccaaataat gattttattt tgactgatag tgacctgttc gttgcaacaa attgataagc
      481 aatgcttttt tataatgcca actttgtaca aaaaagcagg ctttttaatt aacgacttta
      541 ggtccaagct tccctcatga gcttactaaa agccagataa cagtatgcgt atttgcgcgc
      601 tgatttttgc ggtataagaa tatatactga tatgtatacc cgaagtatgt caaaaagagg
      661 tgtgcttcta gaatgcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg
      721 tttgtggatg tacagagtga tattattgac acgcccgggc gacggatagt gatccccctg
      781 gccagtgcac gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc
      841 ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc
      901 ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg
      961 atgttctggg gaatatagtg ctgatatcga cccaagtacc gccacctaac aattcgttca
     1021 agccgagatc ggcttcccgg ccgcggagtt gttcggtaaa ttgtcacaac gccgcccgcc
     1081 ccgttccact gagcgtcaga cccggtagaa aagatcaaag gatcttcttg agatcctttt
     1141 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     1201 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     1261 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     1321 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     1381 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     1441 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     1501 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     1561 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     1621 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     1681 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     1741 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     1801 tctgtggata accgtattac cgctagcatg gatctcgggg acgtctaact actaagcgag
     1861 agtagggaac tgccaggcat caaataaaac gaaaggctca gtcggaagac tgggcctttc
     1921 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg
     1981 atttgaacgt tgtgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg
     2041 ccaggcatca aactaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa
     2101 ctcttcctgt tagttagtta cttaaggcta gcggaattcg acctaaagtc ggcgcgccag
     2161 ctagacccag ctttcttgta caaagttggc attataagaa agcattgctt atcaatttgt
     2221 tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttgccatcc agctgcaggc
     2281 cctatagtga gtcgtattac cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     2341 tccgctctca tggcggccgc caatgccaat aggatatcgg cattttcttt tgcgttttta
     2401 tttgttaact gttaattgtc cttgttcaag gatgctgtct ttgacaacag atgttttctt
     2461 gcctttgatg ttcagcagga agctaggcgc aaacgttgat tgtttgtctg cgtagaatcc
     2521 tctgtttgtc atatagcttg taatcacgac attgtttcct ttcgcttgag gtacagcgaa
     2581 gtgtgagtaa gtaaaggtta catcgttagg atcaagatcc atttttaaca caaggccagt
     2641 tttgttcagc ggcttgtatg ggccagttaa agaattagaa acataaccaa gcatgtaaat
     2701 atcgttagac gtaatgccgt caatcgtcat ttttgatccg cgcgagtcag tgaacaagta
     2761 ccatttgccg ttcattttaa agacgttcgc gcgttcaatt tcatctgtta ctgtgttaga
     2821 tgcaatcagc ggtttcatca cttttttcag tgtgtaatca tcgtttagct caatcatacc
     2881 gagagcgccg tttgctaact cagccgtgcg ttttttatcg ctttgcagaa gtttttgact
     2941 ttcttgacgg aagaatgatg tgcttttgcc atagtatgct ttgttaaata aagattcttc
     3001 gccttggtag ccatcttcag ttccagtgtt tgcttcaaat actaagtatt tgtggccttt
     3061 atcttctacg tagtgaggat ctctcagcgt atggttgtcg cctgagctgt agttgccttc
     3121 atcgatgaac tgctgtacat tttgatacgt ttttccgtca ccgtcaaaga ttgatttata
     3181 atcctctaca ccgttgatgt tcaaagagct gtctgatgct gatacgttaa cttgtgcagt
     3241 tgtcagtgtt tgtttgccgt aatgtttacc ggagaaatca gtgtagaata aacggatttt
     3301 tccgtcagat gtaaatgtgg ctgaacctga ccattcttgt gtttggtctt ttaggataga
     3361 atcatttgca tcgaatttgt cgctgtcttt aaagacgcgg ccagcgtttt tccagctgtc
     3421 aatagaagtt tcgccgactt tttgatagaa catgtaaatc gatgtgtcat ccgcattttt
     3481 aggatctccg gctaatgcaa agacgatgtg gtagccgtga tagtttgcga cagtgccgtc
     3541 agcgttttgt aatggccagc tgtcccaaac gtccaggcct tttgcagaag agatattttt
     3601 aattgtggac gaatcgaact caggaacttg atatttttca tttttttgct gttcagggat
     3661 ttgcagcata tcatggcgtg taatatggga aatgccgtat gtttccttat atggcttttg
     3721 gttcgtttct ttcgcaaacg cttgagttgc gcctcctgcc agcagtgcgg tagtaaaggt
     3781 taatactgtt gcttgttttg caaacttttt gatgttcatc gttcatgtct ccttttttat
     3841 gtactgtgtt agcggtctgc ttcttccagc cctcctgttt gaagatggca agttagttac
     3901 gcacaataaa aaaagaccta aaatatgtaa ggggtgacgc caaagtatac actttgccct
     3961 ttacacattt taggtcttgc ctgctttatc agtaacaaac ccgcgcgatt tacttttcga
     4021 cctcattcta ttagactctc gtttggattg caactggtct attttcctct tttgtttgat
     4081 agaaaatcat aaaaggattt gcagactacg ggcctaaaga actaaaaaat ctatctgttt
     4141 cttttcattc tctgtatttt ttatagtttc tgttgcatgg gcataaagtt gcctttttaa
     4201 tcacaattca gaaaatatca taatatctca tttcactaaa taatagtgaa cggcaggtat
     4261 atgtggtcga gcctgatagt ttggctgtga gcaattatgt gcttagtgca tctaatcgct
     4321 tgagttaacg ccggcgaagc ggcgtcggct tgaacgaatt tctagctaga ggatcgcacc
     4381 aataactgcc ttaaaaaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt
     4441 cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca
     4501 gcggcatcag caccttgtcg ccttgcgtat aatatttgcc cattgtgaaa acgggggcga
     4561 agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg
     4621 ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt
     4681 aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac
     4741 tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac
     4801 tatcccatat caccagctca ccgtctttca ttgccatacg gaactccgga tgagcattca
     4861 tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg
     4921 tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg
     4981 actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc
     5041 cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgac gatcctaact
     5101 caaaatccac acattatacg agccggaagc ataaagtgta aagcctgggg gtgcctaatg
     5161 cgtctagctg cgttacatcc ctggcttgtt gtccacaacc gttaaacctt aacagcttta
     5221 aaagccttat atattctttt ttttcttata aaacttaaaa ccttagaggc tatttaagtt
     5281 gctgatttat attaatttta ttgttcaaac atgagagctt agtacgtgaa acatgagagc
     5341 ttagtacgtt agccatgaga gcttagtacg ttagccatga gggtttagtt cgttaaacat
     5401 gagagcttag tacgttaaac atgagagctt agtacgtgaa acatgagagc ttagtacgta
     5461 ctatcaacag gttgaactgc tgatcttcag atcctctacg ccggacgcat cgtggccgga
     5521 tcaattccgt tttccgctgc ataaccctgc ttcggggtca ttatagcgat tttttcggta
     5581 tatccatcct ttttcgcacg atatacagga ttttgccaaa gggttcgtgt agactttcct
     5641 tggtgtatcc aacggcgtca gccgggcagg ataggtgaag taggcccacc cgcgagcggg
     5701 tgttccttct tcactgtccc ttattcgcac ctggcggtgc tcaacgggaa tcctgctctg
     5761 cgaggctggc cggctaccgc cggcgtaaca gatgagggca agcggatggc tgatgaaacc
     5821 aagccaacca ggaagggcag cccacctatc atcatgg
//
