LOCUS       sequence-385918-        4588 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing the sacB gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4588)
  AUTHORS   Li C, Wang L, Cseke LJ, Vasconcelos F, Huguet-Tapia JC, Gassmann W, 
            Pauwels L, White FF, Dong H, Yang B
  TITLE     Efficient CRISPR-Cas9 based cytosine base editors for 
            phytopathogenic bacteria.
  JOURNAL   Commun Biol. 2023 Jan 17;6(1):56. doi: 10.1038/s42003-023-04451-8.
  PUBMED    36646768
REFERENCE   2  (bases 1 to 4588)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Commun 
            Biol."; date: "2023-01-17"; volume: "6(1):56. doi"; pages: " 
            10.1038/s42003-023-04451-8"
COMMENT     Sequence Label: pEN-sacB
FEATURES             Location/Qualifiers
     source          1..4588
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..35
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     27..44
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     28..44
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(115..238)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        280..725
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(726..812,813..2147)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     protein_bind    complement(2188..2283)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2320..2339)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2321..2339)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2344..2360)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2344..2360)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2462..2481)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2473..3282
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2540..2559)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3429..4017
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3918..3937
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4171..4188
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4347..4374)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4466..4552)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 catggatgtt ttcccagtca cgacgttgta aaacgacggc cagtcttaag ctcgggccct
       61 gcagctctag agctcgaatt ctacaggtca ctaataccat ctaagtagtt ggttcatagt
      121 gactgcatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt
      181 aatatattga tatttatatc attttacgtt tctcgttcaa ctttcttgta caaagtggct
      241 actataagct aggtagtaac tagtacctgg atgcaggtgc acatatacct gccgttcact
      301 attatttagt gaaatgagat attatgatat tttctgaatt gtgattaaaa aggcaacttt
      361 atgcccatgc aacagaaact ataaaaaata cagagaatga aaagaaacag atagattttt
      421 tagttcttta ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa caaaagagga
      481 aaatagacca gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa agtaaatcgc
      541 gcgggtttgt tactgataaa gcaggcaaga cctaaaatgt gtaaagggca aagtgtatac
      601 tttggcgtca ccccttacat attttaggtc tttttttatt gtgcgtaact aacttgccat
      661 cttcaaacag gagggctgga agaagcagac cgctaacaca gtacataaaa aaggagacat
      721 gaacgatgaa catcaaaaag tttgcaaaac aagcaacagt attaaccttt actaccgcac
      781 tgctggcagg aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag ccatataagg
      841 aaacatacgg catttcccat attacacgcc atgatatgct gcaaatccct gaacagcaaa
      901 aaaatgaaaa atatcaagtt cctgagttcg attcgtccac aattaaaaat atctcttctg
      961 caaaaggcct ggacgtttgg gacagctggc cattacaaaa cgctgacggc actgtcgcaa
     1021 actatcacgg ctaccacatc gtctttgcat tagccggaga tcctaaaaat gcggatgaca
     1081 catcgattta catgttctat caaaaagtcg gcgaaacttc tattgacagc tggaaaaacg
     1141 ctggccgcgt ctttaaagac agcgacaaat tcgatgcaaa tgattctatc ctaaaagacc
     1201 aaacacaaga atggtcaggt tcagccacat ttacatctga cggaaaaatc cgtttattct
     1261 acactgattt ctccggtaaa cattacggca aacaaacact gacaactgca caagttaacg
     1321 tatcagcatc agacagctct ttgaacatca acggtgtaga ggattataaa tcaatctttg
     1381 acggtgacgg aaaaacgtat caaaatgtac agcagttcat cgatgaaggc aactacagct
     1441 caggcgacaa ccatacgctg agagatcctc actacgtaga agataaaggc cacaaatact
     1501 tagtatttga agcaaacact ggaactgaag atggctacca aggcgaagaa tctttattta
     1561 acaaagcata ctatggcaaa agcacatcat tcttccgtca agaaagtcaa aaacttctgc
     1621 aaagcgataa aaaacgcacg gctgagttag caaacggcgc tctcggtatg attgagctaa
     1681 acgatgatta cacactgaaa aaagtgatga aaccgctgat tgcatctaac acagtaacag
     1741 atgaaattga acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg ttcactgact
     1801 cccgcggatc aaaaatgacg attgacggca ttacgtctaa cgatatttac atgcttggtt
     1861 atgtttctaa ttctttaact ggcccataca agccgctgaa caaaactggc cttgtgttaa
     1921 aaatggatct tgatcctaac gatgtaacct ttacttactc acacttcgct gtacctcaag
     1981 cgaaaggaaa caatgtcgtg attacaagct atatgacaaa cagaggattc tacgcagaca
     2041 aacaatcaac gtttgcgccg agcttcctgc tgaacatcaa aggcaagaaa acatctgttg
     2101 tcaaagacag catccttgaa caaggacaat taacagttaa caaataacac ctgcagctgt
     2161 atctagaatc gcgtatgcga gtgtatgcaa ctttattata caaagttggc attataaaaa
     2221 agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta
     2281 tttggagctc catggtagcg ttaacgcggc cgcgatatcc cctatagtga gtcgtattac
     2341 atggtcatag ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg atgttacatt
     2401 gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat
     2461 acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt aaattccaac
     2521 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     2581 acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     2641 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     2701 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     2761 actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     2821 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     2881 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     2941 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     3001 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     3061 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     3121 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     3181 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     3241 aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt ggttaattgg
     3301 ttgtaacact ggcagagcat tacgctgact tgacgggacg gcgcaagctc atgaccaaaa
     3361 tcccttaacg tgagttacgc gtcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3421 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3481 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3541 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     3601 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3661 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3721 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3781 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3841 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3901 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3961 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4021 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4081 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4141 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4201 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     4261 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatac gcgtaccgct
     4321 agccaggaag agtttgtaga aacgcaaaaa ggccatccgt caggatggcc ttctgcttag
     4381 tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca
     4441 caacgttcaa atccgctccc ggcggatttg tcctactcag gagagcgttc accgacaaac
     4501 aacagataaa acgaaaggcc cagacttccg actgagcctt tcgttttatt tgatgcctgg
     4561 cagttcccta ctctcgcgtt aacgctag
//
