LOCUS       sequence-273773-        4500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration and excision of chloromaphenicol 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 4500)
  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 4500)
  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: pBDC
FEATURES             Location/Qualifiers
     source          1..4500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        30..132
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             133..792
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(199..218)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     complement(1041..1057)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1041..1057)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1058..1076)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             join(1388..1474,1475..2809)
                     /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      3123..3668
                     /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     3569..3588
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3785..3802
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4194..4284
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     promoter        4291..4309
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4311..4328
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4312..4328
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tgggccaact tttggcgaaa atgagacgtt gatcggcacg taagaggttc caactttcac
       61 cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt ttcaggagct
      121 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg
      181 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc
      241 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat
      301 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggagtt ccgtatggca
      361 atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat
      421 gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt
      481 ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa
      541 gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt caccagtttt
      601 gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcactat gggcaaatat
      661 tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt
      721 gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag
      781 ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg ttgctacgcc
      841 tgaataagtg ataataagcg gatgaatggc agaaattcga aagcaaattc gacccggtcg
      901 tcggttcagg gcagggtcgt taaatagccg cttatgtcta ttgctggtag ggataacagg
      961 gtaattctcc ctatagtgag tcgtaggatc cccatggctg caggcatgcc tcgagaccaa
     1021 actgtctcac gacgttttgg gtcatagctg tttcctgccg ctgagcaata actagcgtcg
     1081 actagttctt taggcccgta gtctgcaaat ccttttatga ttttctatca aacaaaagag
     1141 gaaaatagac cagttgcaat ccaaacgaga gtctaataga atgaggtcga aaagtaaatc
     1201 gcgcgggttt gttactgata aagcaggcaa gacctaaaat gtgtaaaggg caaagtgtat
     1261 actttggcgt caccccttac atattttagg tcttttttta ttgtgcgtaa ctaacttgcc
     1321 atcttcaaac aggagggctg gaagaagcag accgctaaca cagtacataa aaaaggagac
     1381 atgaacgatg aacatcaaaa agtttgcaaa acaagcaaca gtattaacct ttactaccgc
     1441 actgctggca ggaggcgcaa ctcaagcgtt tgcgaaagaa acgaaccaaa agccatataa
     1501 ggaaacatac ggcatttccc atattacacg ccatgatatg ctgcaaatcc ctgaacagca
     1561 aaaaaatgaa aaatatcaag ttcctgaatt tgattcgtcc acaattaaaa atatctcttc
     1621 tgcaaaaggc ctggacgttt gggacagctg gccattacaa aacgctgacg gcactgtcgc
     1681 aaactatcac ggctaccaca tcgtctttgc attagccgga gatcctaaaa atgcggatga
     1741 cacatcgatt tacatgttct atcaaaaagt cggcgaaact tctattgaca gctggaaaaa
     1801 cgctggccgc gtctttaaag acagcgacaa attcgatgca aatgattcta tcctaaaaga
     1861 ccaaacacaa gaatggtcag gttcagccac atttacatct gacggaaaaa tccgtttatt
     1921 ctacactgat ttctccggta aacattacgg caaacaaaca ctgacaactg cacaagttaa
     1981 cgtatcagca tcagacagct ctttgaacat caacggtgta gaggattata aatcaatctt
     2041 tgacggtgac ggaaaaacgt atcaaaatgt acagcagttc atcgatgaag gcaactacag
     2101 ctcaggcgac aaccatacgc tgagagatcc tcactacgta gaagataaag gccacaaata
     2161 cttagtattt gaagcaaaca ctggaactga agatggctac caaggcgaag aatctttatt
     2221 taacaaagca tactatggca aaagcacatc attcttccgt caagaaagtc aaaaacttct
     2281 gcaaagcgat aaaaaacgca cggctgagtt agcaaacggc gctctcggta tgattgagct
     2341 aaacgatgat tacacactga aaaaagtgat gaaaccgctg attgcatcta acacagtaac
     2401 agatgaaatt gaacgcgcga acgtctttaa aatgaacggc aaatggtacc tgttcactga
     2461 ctcccgcgga tcaaaaatga cgattgacgg cattacgtct aacgatattt acatgcttgg
     2521 ttatgtttct aattctttaa ctggcccata caagccgctg aacaaaactg gccttgtgtt
     2581 aaaaatggat cttgatccta acgatgtaac ctttacttac tcacacttcg ctgtacctca
     2641 agcgaaagga aacaatgtcg tgattacaag ctatatgaca aacagaggat tctacgcaga
     2701 caaacaatca acgtttgcgc ctagcttcct gctgaacatc aaaggcaaga aaacatctgt
     2761 tgtcaaagac agcatccttg aacaaggaca attaacagtt aacaaataaa aacgcaaaag
     2821 aaaatgccga tatcctattg gcattttctt ttatttctta tcaacataaa ggtgaatccc
     2881 atagaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc
     2941 gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg
     3001 gggcatgact aacatgagaa ttacaactta tatcgtatgg ggctgacttc aggtgctaca
     3061 tttgaagaga taaattgcac tgaaatctag aaatatttta tctgattaat aagatgatct
     3121 tcttgagatc gttttggtct gcgcgtaatc tcttgctctg aaaacgaaaa aaccgccttg
     3181 cagggcggtt tttcgaaggt tctctgagct accaactctt tgaaccgagg taactggctt
     3241 ggaggagcgc agtcaccaaa acttgtcctt tcagtttagc cttaaccggc gcatgacttc
     3301 aagactaact cctctaaatc aattaccagt ggctgctgcc agtggtgctt ttgcatgtct
     3361 ttccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg actgaacggg
     3421 gggttcgtgc atacagtcca gcttggagcg aactgcctac ccggaactga gtgtcaggcg
     3481 tggaatgaga caaacgcggc cataacagcg gaatgacacc ggtaaaccga aaggcaggaa
     3541 caggagagcg cacgagggag ccgccagggg gaaacgcctg gtatctttat agtcctgtcg
     3601 ggtttcgcca ccactgattt gagcgtcaga tttcgtgatg cttgtcaggg gggcggagcc
     3661 tatggaaaaa cggctttgcc gcggccctct cacttccctg ttaagtatct tcctggcatc
     3721 ttccaggaaa tctccgcccc gttcgtaagc catttccgct cgccgcagtc gaacgaccga
     3781 gcgtagcgag tcagtgagcg aggaagcgga atatatcctg tatcacatat tctgctgacg
     3841 caccggtgca gccttttttc tcctgccaca tgaagcactt cactgacacc ctcatcagtg
     3901 ccaacatagt aagccagtat acactccgct agcgctgatg tccggcggtg cttttgccgt
     3961 tacgcaccac cccgtcagta gctgaacagg agggacagct gatagaaaca gaagccactg
     4021 gagcacctca aaaacaccat catacactaa atcagtaagt tggcagcatc acccgacgca
     4081 ctttgcgccg aataaatacc tgtgacggaa gatcacttcg cagaataaat aaatcctggt
     4141 gtccctgttg ataccgggaa gccctgggcc aacttttggc gaaaatgaga cgttgatcgg
     4201 cacgtaagag gttccaactt tcaccataat gaaataagat cactaccggg cgtatttttt
     4261 gagttatcga gattttcagg agctagatct taatacgact cactataggg tgtaaaacga
     4321 cggccagtca aaacgtcgtg agacagtttg gtaagcttgg tacccaattg gagctcacta
     4381 gtgaattcgt agcacacgag gtctcttagg gataacaggg taatcgaata aatacctgtg
     4441 acggaagatc acttcgcaga ataaataaat cctggtgtcc ctgttgatac cgggaagccc
//
