LOCUS       sequence-260073-        4604 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration and excision of kanamycin cassette for the E. coli 
            genome via CreI, SceI, and lamba Red system. Contains SacB 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4604)
  AUTHORS   Wei Y, Deng P, Mohsin A, Yang Y, Zhou H, Guo M, Fang H
  TITLE     An electroporation-free method based on Red recombineering for 
            markerless deletion and genomic replacement in the Escherichia coli 
            DH1 genome.
  JOURNAL   PLoS One. 2017 Oct 24;12(10):e0186891. doi: 
            10.1371/journal.pone.0186891. eCollection 2017.
  PUBMED    29065183
REFERENCE   2  (bases 1 to 4604)
  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: "PLoS One.";
            date: "2017-10-24"; volume: "12(10):e0186891. doi"; pages: " 
            10.1371/journal.pone.0186891. eCollection 2017"
COMMENT     Sequence Label: pBDK
FEATURES             Location/Qualifiers
     source          1..4604
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(277..296)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             288..1103
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             288..1103
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(361..380)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(1289..1305)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1289..1305)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1306..1324)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             join(1636..1722,1723..3057)
                     /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"
     rep_origin      3371..3916
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     3817..3836
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4033..4050
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4442..4532
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     promoter        4539..4557
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4559..4576
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4560..4576
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ttggtaccca attggagctc actagtgaat tcgtagcaca cgaggtctct tagggataac
       61 agggtaatga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
      121 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
      181 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
      241 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
      301 aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg
      361 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg
      421 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
      481 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
      541 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa
      601 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
      661 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
      721 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
      781 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac
      841 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
      901 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
      961 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     1021 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     1081 tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt
     1141 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgtaggg
     1201 ataacagggt aattctccct atagtgagtc gtaggatccc catggctgca ggcatgcctc
     1261 gagaccaaac tgtctcacga cgttttgggt catagctgtt tcctgccgct gagcaataac
     1321 tagcgtcgac tagttcttta ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa
     1381 caaaagagga aaatagacca gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa
     1441 agtaaatcgc gcgggtttgt tactgataaa gcaggcaaga cctaaaatgt gtaaagggca
     1501 aagtgtatac tttggcgtca ccccttacat attttaggtc tttttttatt gtgcgtaact
     1561 aacttgccat cttcaaacag gagggctgga agaagcagac cgctaacaca gtacataaaa
     1621 aaggagacat gaacgatgaa catcaaaaag tttgcaaaac aagcaacagt attaaccttt
     1681 actaccgcac tgctggcagg aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag
     1741 ccatataagg aaacatacgg catttcccat attacacgcc atgatatgct gcaaatccct
     1801 gaacagcaaa aaaatgaaaa atatcaagtt cctgaatttg attcgtccac aattaaaaat
     1861 atctcttctg caaaaggcct ggacgtttgg gacagctggc cattacaaaa cgctgacggc
     1921 actgtcgcaa actatcacgg ctaccacatc gtctttgcat tagccggaga tcctaaaaat
     1981 gcggatgaca catcgattta catgttctat caaaaagtcg gcgaaacttc tattgacagc
     2041 tggaaaaacg ctggccgcgt ctttaaagac agcgacaaat tcgatgcaaa tgattctatc
     2101 ctaaaagacc aaacacaaga atggtcaggt tcagccacat ttacatctga cggaaaaatc
     2161 cgtttattct acactgattt ctccggtaaa cattacggca aacaaacact gacaactgca
     2221 caagttaacg tatcagcatc agacagctct ttgaacatca acggtgtaga ggattataaa
     2281 tcaatctttg acggtgacgg aaaaacgtat caaaatgtac agcagttcat cgatgaaggc
     2341 aactacagct caggcgacaa ccatacgctg agagatcctc actacgtaga agataaaggc
     2401 cacaaatact tagtatttga agcaaacact ggaactgaag atggctacca aggcgaagaa
     2461 tctttattta acaaagcata ctatggcaaa agcacatcat tcttccgtca agaaagtcaa
     2521 aaacttctgc aaagcgataa aaaacgcacg gctgagttag caaacggcgc tctcggtatg
     2581 attgagctaa acgatgatta cacactgaaa aaagtgatga aaccgctgat tgcatctaac
     2641 acagtaacag atgaaattga acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg
     2701 ttcactgact cccgcggatc aaaaatgacg attgacggca ttacgtctaa cgatatttac
     2761 atgcttggtt atgtttctaa ttctttaact ggcccataca agccgctgaa caaaactggc
     2821 cttgtgttaa aaatggatct tgatcctaac gatgtaacct ttacttactc acacttcgct
     2881 gtacctcaag cgaaaggaaa caatgtcgtg attacaagct atatgacaaa cagaggattc
     2941 tacgcagaca aacaatcaac gtttgcgcct agcttcctgc tgaacatcaa aggcaagaaa
     3001 acatctgttg tcaaagacag catccttgaa caaggacaat taacagttaa caaataaaaa
     3061 cgcaaaagaa aatgccgata tcctattggc attttctttt atttcttatc aacataaagg
     3121 tgaatcccat agaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat
     3181 taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg
     3241 tgggcgcggg gcatgactaa catgagaatt acaacttata tcgtatgggg ctgacttcag
     3301 gtgctacatt tgaagagata aattgcactg aaatctagaa atattttatc tgattaataa
     3361 gatgatcttc ttgagatcgt tttggtctgc gcgtaatctc ttgctctgaa aacgaaaaaa
     3421 ccgccttgca gggcggtttt tcgaaggttc tctgagctac caactctttg aaccgaggta
     3481 actggcttgg aggagcgcag tcaccaaaac ttgtcctttc agtttagcct taaccggcgc
     3541 atgacttcaa gactaactcc tctaaatcaa ttaccagtgg ctgctgccag tggtgctttt
     3601 gcatgtcttt ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcggac
     3661 tgaacggggg gttcgtgcat acagtccagc ttggagcgaa ctgcctaccc ggaactgagt
     3721 gtcaggcgtg gaatgagaca aacgcggcca taacagcgga atgacaccgg taaaccgaaa
     3781 ggcaggaaca ggagagcgca cgagggagcc gccaggggga aacgcctggt atctttatag
     3841 tcctgtcggg tttcgccacc actgatttga gcgtcagatt tcgtgatgct tgtcaggggg
     3901 gcggagccta tggaaaaacg gctttgccgc ggccctctca cttccctgtt aagtatcttc
     3961 ctggcatctt ccaggaaatc tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga
     4021 acgaccgagc gtagcgagtc agtgagcgag gaagcggaat atatcctgta tcacatattc
     4081 tgctgacgca ccggtgcagc cttttttctc ctgccacatg aagcacttca ctgacaccct
     4141 catcagtgcc aacatagtaa gccagtatac actccgctag cgctgatgtc cggcggtgct
     4201 tttgccgtta cgcaccaccc cgtcagtagc tgaacaggag ggacagctga tagaaacaga
     4261 agccactgga gcacctcaaa aacaccatca tacactaaat cagtaagttg gcagcatcac
     4321 ccgacgcact ttgcgccgaa taaatacctg tgacggaaga tcacttcgca gaataaataa
     4381 atcctggtgt ccctgttgat accgggaagc cctgggccaa cttttggcga aaatgagacg
     4441 ttgatcggca cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg
     4501 tattttttga gttatcgaga ttttcaggag ctagatctta atacgactca ctatagggtg
     4561 taaaacgacg gccagtcaaa acgtcgtgag acagtttggt aagc
//
