LOCUS       sequence-99454-e        5367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Sucrose counterselection (sacB) vector for unmarked allelic 
            exchange..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5367)
  AUTHORS   Puri AW, Owen S, Chu F, Chavkin T, Beck DA, Kalyuzhnaya MG, Lidstrom
            ME
  TITLE     Genetic tools for the industrially promising methanotroph 
            Methylomicrobium buryatense.
  JOURNAL   Appl Environ Microbiol. 2014 Dec 29. pii: AEM.03795-14.
  PUBMED    25548049
REFERENCE   2  (bases 1 to 5367)
  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: "Appl 
            Environ Microbiol. 2014 Dec 29. pii: AEM.03795-14."
COMMENT     Sequence Label: pCM433kanT
FEATURES             Location/Qualifiers
     source          1..5367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(43..631)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(123..142)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1192..1563)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            1596..1705
                     /note="incP origin of transfer"
     primer_bind     complement(2152..2171)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2163..2978
                     /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             2163..2978
                     /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(2236..2255)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(join(3131..4465,4466..4552))
                     /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"
     promoter        complement(4553..4998)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     primer_bind     complement(5239..5256)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
       61 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
      121 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
      181 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
      241 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
      301 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
      361 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
      421 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
      481 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
      541 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
      601 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
      661 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
      721 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
      781 tgagtaaact tggtctgaca gttaccaatg tgcaggttgt cggtgtcgtg atgcacggca
      841 ctgacgcgct gatgctcggc gaagccaagc ccagcgcaga tgcggtcctc aatcgcgcgc
      901 aacgtctccg cgtcgggctt ctctcccgcg cggaagctaa ccagcaggtg ataggtcttg
      961 tcggcctcgg aacgggtgtt gccgtgctgg gtcgccatca cctcggccat gacagcgggc
     1021 agggtgtttg cctcgcagtt cgtgacgcgc acgtgaccca ggcgctcggt cttgccttgc
     1081 tcgtcggtga tgtacttcac cagctccgcg aagtcgctct tcttgatgga gcgcatgggg
     1141 acgtgcttgg caatcacgcg caccccccgg ccgttttagc ggctaaaaaa gtcatggctc
     1201 tgccctcggg cggaccacgc ccatcatgac cttgccaagc tcgtcctgct tctcttcgat
     1261 cttcgccagc agggcgagga tcgtggcatc accgaaccgc gccgtgcgcg ggtcgtcggt
     1321 gagccagagt ttcagcaggc cgcccaggcg gcccaggtcg ccattgatgc gggccagctc
     1381 gcggacgtgc tcatagtcca cgacgcccgt gattttgtag ccctggccga cggccagcag
     1441 gtaggccgac aggctcatgc cggccgccgc cgccttttcc tcaatcgctc ttcgttcgtc
     1501 tggaaggcag tacaccttga taggtgggct gcccttcctg gttggcttgg tttcatcagc
     1561 catccgcttg ccctcatctg ttacgccggc ggtagccggc cagcctcgca gagcaggatt
     1621 cccgttgagc accgccaggt gcgaataagg gacagtgaag aaggaacacc cgctcgcggg
     1681 tgggcctact tcacctatcc tgcccggctg acgccgttgg atacaccaag gaaagtctac
     1741 acgaaccctt tggcaaaatc ctgtatatcg tgcgaaaaag gatggatata ccgaaaaaat
     1801 cgctataatg accccgaagc agggttatgc agcggaaaag cgctgcttcc ctgctgtttt
     1861 gtggaatatc taccgactgg aaacaggcaa atgcaggaaa ttactgaact gaggggacag
     1921 ggttgatgag agctttgttg taggtggacc agttggtgat tttgaacttt tgctttgcca
     1981 cggaacggtc tgcgttgtcg ggaagatgcg tgatctgatc cttcaactca gcaaaagttc
     2041 gatttattca acaaagccac gttgtgtctc aaaatctctg atgttacatt gcacaagata
     2101 aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg
     2161 ttatgagcca tattcaacgg gaaacgtctt gctcgaggcc gcgattaaat tccaacatgg
     2221 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     2281 tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
     2341 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     2401 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     2461 cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     2521 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     2581 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt
     2641 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     2701 aagaaatgca taagcttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     2761 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
     2821 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     2881 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     2941 aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt
     3001 aacactggca gagcattacg ctgacttgac gggactcgag attatagctc ctgccctatg
     3061 ggattcacct ttatgttgat aagaaataaa agaaaatgcc aataggatat cggcattttc
     3121 ttttgcgttt ttatttgtta actgttaatt gtccttgttc aaggatgctg tctttgacaa
     3181 cagatgtttt cttgcctttg atgttcagca ggaagcttgg cgcaaacgtt gattgtttgt
     3241 ctgcgtagaa tcctctgttt gtcatatagc ttgtaatcac gacattgttt cctttcgctt
     3301 gaggtacagc gaagtgtgag taagtaaagg ttacatcgtt aggatcaaga tccattttta
     3361 acacaaggcc agttttgttc agcggcttgt atgggccagt taaagaatta gaaacataac
     3421 caagcatgta aatatcgtta gacgtaatgc cgtcaatcgt catttttgat ccgcgggagt
     3481 cagtgaacag gtaccatttg ccgttcattt taaagacgtt cgcgcgttca atttcatctg
     3541 ttactgtgtt agatgcaatc agcggtttca tcactttttt cagtgtgtaa tcatcgttta
     3601 gctcaatcat accgagagcg ccgtttgcta actcagccgt gcgtttttta tcgctttgca
     3661 gaagtttttg actttcttga cggaagaatg atgtgctttt gccatagtat gctttgttaa
     3721 ataaagattc ttcgccttgg tagccatctt cagttccagt gtttgcttca aatactaagt
     3781 atttgtggcc tttatcttct acgtagtgag gatctctcag cgtatggttg tcgcctgagc
     3841 tgtagttgcc ttcatcgatg aactgctgta cattttgata cgtttttccg tcaccgtcaa
     3901 agattgattt ataatcctct acaccgttga tgttcaaaga gctgtctgat gctgatacgt
     3961 taacttgtgc agttgtcagt gtttgtttgc cgtaatgttt accggagaaa tcagtgtaga
     4021 ataaacggat ttttccgtca gatgtaaatg tggctgaacc tgaccattct tgtgtttggt
     4081 cttttaggat agaatcattt gcatcgaatt tgtcgctgtc tttaaagacg cggccagcgt
     4141 ttttccagct gtcaatagaa gtttcgccga ctttttgata gaacatgtaa atcgatgtgt
     4201 catccgcatt tttaggatct ccggctaatg caaagacgat gtggtagccg tgatagtttg
     4261 cgacagtgcc gtcagcgttt tgtaatggcc agctgtccca aacgtccagg ccttttgcag
     4321 aagagatatt tttaattgtg gacgaatcga attcaggaac ttgatatttt tcattttttt
     4381 gctgttcagg gatttgcagc atatcatggc gtgtaatatg ggaaatgccg tatgtttcct
     4441 tatatggctt ttggttcgtt tctttcgcaa acgcttgagt tgcgcctcct gccagcagtg
     4501 cggtagtaaa ggttaatact gttgcttgtt ttgcaaactt tttgatgttc atcgttcatg
     4561 tctccttttt tatgtactgt gttagcggtc tgcttcttcc agccctcctg tttgaagatg
     4621 gcaagttagt tacgcacaat aaaaaaagac ctaaaatatg taaggggtga cgccaaagta
     4681 tacactttgc cctttacaca ttttaggtct tgcctgcttt atcagtaaca aacccgcgcg
     4741 atttactttt cgacctcatt ctattagact ctcgtttgga ttgcaactgg tctattttcc
     4801 tcttttgttt gatagaaaat cataaaagga tttgcagact acgggcctaa agaactaaaa
     4861 aatctatctg tttcttttca ttctctgtat tttttatagt ttctgttgca tgggcataaa
     4921 gttgcctttt taatcacaat tcagaaaata tcataatatc tcatttcact aaataatagt
     4981 gaacggcagg tatatgtgat gggttaaaaa ggatcgatcc tctagccacg ggtgcgcatg
     5041 atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag
     5101 aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc
     5161 tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag
     5221 tcagcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5281 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5341 aggaaagaac atgtgagcaa aaggcca
//
