LOCUS       sequence-260075-        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"
     primer_bind     complement(26..43)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(26..42)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(45..63)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(70..160)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(552..569)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(686..1231)
                     /direction=LEFT
                     /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     complement(766..785)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1545..2879,2880..2966))
                     /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"
     primer_bind     3278..3296
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     primer_bind     3297..3313
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3297..3313
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(3562..4221)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4136..4155
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4222..4324)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 taccaaactg tctcacgacg ttttgactgg ccgtcgtttt acaccctata gtgagtcgta
       61 ttaagatcta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat
      121 ttcattatgg tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccaaaa
      181 gttggcccag ggcttcccgg tatcaacagg gacaccagga tttatttatt ctgcgaagtg
      241 atcttccgtc acaggtattt attcggcgca aagtgcgtcg ggtgatgctg ccaacttact
      301 gatttagtgt atgatggtgt ttttgaggtg ctccagtggc ttctgtttct atcagctgtc
      361 cctcctgttc agctactgac ggggtggtgc gtaacggcaa aagcaccgcc ggacatcagc
      421 gctagcggag tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct
      481 tcatgtggca ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata
      541 tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa
      601 atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa
      661 gtgagagggc cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg
      721 aaatctgacg ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt
      781 ttccccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca
      841 ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca
      901 gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta
      961 tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca
     1021 gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac
     1081 tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt
     1141 ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca
     1201 agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc agataaaata
     1261 tttctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg
     1321 atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga gctgactggg
     1381 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     1441 ttaattgcgt tgcgctcact gcccgctttc tatgggattc acctttatgt tgataagaaa
     1501 taaaagaaaa tgccaatagg atatcggcat tttcttttgc gtttttattt gttaactgtt
     1561 aattgtcctt gttcaaggat gctgtctttg acaacagatg ttttcttgcc tttgatgttc
     1621 agcaggaagc taggcgcaaa cgttgattgt ttgtctgcgt agaatcctct gtttgtcata
     1681 tagcttgtaa tcacgacatt gtttcctttc gcttgaggta cagcgaagtg tgagtaagta
     1741 aaggttacat cgttaggatc aagatccatt tttaacacaa ggccagtttt gttcagcggc
     1801 ttgtatgggc cagttaaaga attagaaaca taaccaagca tgtaaatatc gttagacgta
     1861 atgccgtcaa tcgtcatttt tgatccgcgg gagtcagtga acaggtacca tttgccgttc
     1921 attttaaaga cgttcgcgcg ttcaatttca tctgttactg tgttagatgc aatcagcggt
     1981 ttcatcactt ttttcagtgt gtaatcatcg tttagctcaa tcataccgag agcgccgttt
     2041 gctaactcag ccgtgcgttt tttatcgctt tgcagaagtt tttgactttc ttgacggaag
     2101 aatgatgtgc ttttgccata gtatgctttg ttaaataaag attcttcgcc ttggtagcca
     2161 tcttcagttc cagtgtttgc ttcaaatact aagtatttgt ggcctttatc ttctacgtag
     2221 tgaggatctc tcagcgtatg gttgtcgcct gagctgtagt tgccttcatc gatgaactgc
     2281 tgtacatttt gatacgtttt tccgtcaccg tcaaagattg atttataatc ctctacaccg
     2341 ttgatgttca aagagctgtc tgatgctgat acgttaactt gtgcagttgt cagtgtttgt
     2401 ttgccgtaat gtttaccgga gaaatcagtg tagaataaac ggatttttcc gtcagatgta
     2461 aatgtggctg aacctgacca ttcttgtgtt tggtctttta ggatagaatc atttgcatcg
     2521 aatttgtcgc tgtctttaaa gacgcggcca gcgtttttcc agctgtcaat agaagtttcg
     2581 ccgacttttt gatagaacat gtaaatcgat gtgtcatccg catttttagg atctccggct
     2641 aatgcaaaga cgatgtggta gccgtgatag tttgcgacag tgccgtcagc gttttgtaat
     2701 ggccagctgt cccaaacgtc caggcctttt gcagaagaga tatttttaat tgtggacgaa
     2761 tcaaattcag gaacttgata tttttcattt ttttgctgtt cagggatttg cagcatatca
     2821 tggcgtgtaa tatgggaaat gccgtatgtt tccttatatg gcttttggtt cgtttctttc
     2881 gcaaacgctt gagttgcgcc tcctgccagc agtgcggtag taaaggttaa tactgttgct
     2941 tgttttgcaa actttttgat gttcatcgtt catgtctcct tttttatgta ctgtgttagc
     3001 ggtctgcttc ttccagccct cctgtttgaa gatggcaagt tagttacgca caataaaaaa
     3061 agacctaaaa tatgtaaggg gtgacgccaa agtatacact ttgcccttta cacattttag
     3121 gtcttgcctg ctttatcagt aacaaacccg cgcgatttac ttttcgacct cattctatta
     3181 gactctcgtt tggattgcaa ctggtctatt ttcctctttt gtttgataga aaatcataaa
     3241 aggatttgca gactacgggc ctaaagaact agtcgacgct agttattgct cagcggcagg
     3301 aaacagctat gacccaaaac gtcgtgagac agtttggtct cgaggcatgc ctgcagccat
     3361 ggggatccta cgactcacta tagggagaat taccctgtta tccctaccag caatagacat
     3421 aagcggctat ttaacgaccc tgccctgaac cgacgaccgg gtcgaatttg ctttcgaatt
     3481 tctgccattc atccgcttat tatcacttat tcaggcgtag caaccaggcg tttaagggca
     3541 ccaataactg ccttaaaaaa attacgcccc gccctgccac tcatcgcagt actgttgtaa
     3601 ttcattaagc attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc
     3661 cagcggcatc agcaccttgt cgccttgcgt ataatatttg cccatagtga aaacgggggc
     3721 gaagaagttg tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt
     3781 ggctgagacg aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc
     3841 gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc
     3901 actccagagc gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac
     3961 actatcccat atcaccagct caccgtcttt cattgccata cggaactccg gatgagcatt
     4021 catcaggcgg gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac
     4081 ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac
     4141 tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata
     4201 tccagtgatt tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa
     4261 aaatacgccc ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccg
     4321 atcaacgtct cattttcgcc aaaagttggc ccagggcttc ccggtatcaa cagggacacc
     4381 aggatttatt tattctgcga agtgatcttc cgtcacaggt atttattcga ttaccctgtt
     4441 atccctaaga gacctcgtgt gctacgaatt cactagtgag ctccaattgg gtaccaagct
//
