LOCUS       sequence-352793-        8081 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8081)
  AUTHORS   Marx CJ
  TITLE     Development of a broad-host-range sacB-based vector for unmarked 
            allelic exchange.
  JOURNAL   BMC Res Notes. 2008 Feb 26;1:1. doi: 10.1186/1756-0500-1-1.
  PUBMED    18710539
REFERENCE   2  (bases 1 to 8081)
  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: "BMC Res 
            Notes."; date: "2008-02-26"; volume: "1:1. doi"; pages: " 
            10.1186/1756-0500-1-1"
COMMENT     Sequence Label: pCM433
FEATURES             Location/Qualifiers
     source          1..8081
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     240..259
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(join(423..1757,1758..1844))
                     /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(1845..2290)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     primer_bind     complement(2531..2548)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2702..3290)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2782..2801)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3461..4252,4253..4321))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4084..4103
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4322..4426)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(5153..5524)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            5557..5666
                     /note="incP origin of transfer"
     promoter        join(6303..6308,6309..6325,6326..6331)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             6379..7569
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(6731..6750)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(join(7747..8081,1..325))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt
       61 gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaat tccggatgag
      121 cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct
      181 ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag
      241 caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg
      301 tatatccagt gatttttttc tccattttag cttccttagc tcctgcccta tgggattcac
      361 ctttatgttg ataagaaata aaagaaaatg ccaataggat atcggcattt tcttttgcgt
      421 ttttatttgt taactgttaa ttgtccttgt tcaaggatgc tgtctttgac aacagatgtt
      481 ttcttgcctt tgatgttcag caggaagctt ggcgcaaacg ttgattgttt gtctgcgtag
      541 aatcctctgt ttgtcatata gcttgtaatc acgacattgt ttcctttcgc ttgaggtaca
      601 gcgaagtgtg agtaagtaaa ggttacatcg ttaggatcaa gatccatttt taacacaagg
      661 ccagttttgt tcagcggctt gtatgggcca gttaaagaat tagaaacata accaagcatg
      721 taaatatcgt tagacgtaat gccgtcaatc gtcatttttg atccgcggga gtcagtgaac
      781 aggtaccatt tgccgttcat tttaaagacg ttcgcgcgtt caatttcatc tgttactgtg
      841 ttagatgcaa tcagcggttt catcactttt ttcagtgtgt aatcatcgtt tagctcaatc
      901 ataccgagag cgccgtttgc taactcagcc gtgcgttttt tatcgctttg cagaagtttt
      961 tgactttctt gacggaagaa tgatgtgctt ttgccatagt atgctttgtt aaataaagat
     1021 tcttcgcctt ggtagccatc ttcagttcca gtgtttgctt caaatactaa gtatttgtgg
     1081 cctttatctt ctacgtagtg aggatctctc agcgtatggt tgtcgcctga gctgtagttg
     1141 ccttcatcga tgaactgctg tacattttga tacgtttttc cgtcaccgtc aaagattgat
     1201 ttataatcct ctacaccgtt gatgttcaaa gagctgtctg atgctgatac gttaacttgt
     1261 gcagttgtca gtgtttgttt gccgtaatgt ttaccggaga aatcagtgta gaataaacgg
     1321 atttttccgt cagatgtaaa tgtggctgaa cctgaccatt cttgtgtttg gtcttttagg
     1381 atagaatcat ttgcatcgaa tttgtcgctg tctttaaaga cgcggccagc gtttttccag
     1441 ctgtcaatag aagtttcgcc gactttttga tagaacatgt aaatcgatgt gtcatccgca
     1501 tttttaggat ctccggctaa tgcaaagacg atgtggtagc cgtgatagtt tgcgacagtg
     1561 ccgtcagcgt tttgtaatgg ccagctgtcc caaacgtcca ggccttttgc agaagagata
     1621 tttttaattg tggacgaatc gaattcagga acttgatatt tttcattttt ttgctgttca
     1681 gggatttgca gcatatcatg gcgtgtaata tgggaaatgc cgtatgtttc cttatatggc
     1741 ttttggttcg tttctttcgc aaacgcttga gttgcgcctc ctgccagcag tgcggtagta
     1801 aaggttaata ctgttgcttg ttttgcaaac tttttgatgt tcatcgttca tgtctccttt
     1861 tttatgtact gtgttagcgg tctgcttctt ccagccctcc tgtttgaaga tggcaagtta
     1921 gttacgcaca ataaaaaaag acctaaaata tgtaaggggt gacgccaaag tatacacttt
     1981 gccctttaca cattttaggt cttgcctgct ttatcagtaa caaacccgcg cgatttactt
     2041 ttcgacctca ttctattaga ctctcgtttg gattgcaact ggtctatttt cctcttttgt
     2101 ttgatagaaa atcataaaag gatttgcaga ctacgggcct aaagaactaa aaaatctatc
     2161 tgtttctttt cattctctgt attttttata gtttctgttg catgggcata aagttgcctt
     2221 tttaatcaca attcagaaaa tatcataata tctcatttca ctaaataata gtgaacggca
     2281 ggtatatgtg atgggttaaa aaggatcgat cctctagcca cgggtgcgca tgatcgtgct
     2341 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc
     2401 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac
     2461 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgct
     2521 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2581 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2641 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2701 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2761 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2821 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2881 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2941 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3001 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3061 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3121 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3181 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3241 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3301 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3361 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3421 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3481 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3541 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3601 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3661 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3721 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3781 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3841 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3901 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3961 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4021 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4081 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4141 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4201 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4261 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4321 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4381 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4441 aagtgccacc tgacgtctag atctgaattc agctgtacaa ttggtaccat ggatgcatat
     4501 gctgcagctc gagcggccgc gggccctacg tacgcgtgtt aaccggtgag ctcactagag
     4561 gatccagccg accaggcttt ccacgcccgc gtgccgctcc atgtcgttcg cgcggttctc
     4621 ggaaacgcgc tgccgcgttt cgtgattgtc acgctcaagc ccgtagtccc gttcgagcgt
     4681 cgcgcagagg tcagcgaggg cgcggtaggc ccgatacggc tcatggatgg tgtttcgggt
     4741 cgggtgaatc ttgttgatgg cgatatggat gtgcaggttg tcggtgtcgt gatgcacggc
     4801 actgacgcgc tgatgctcgg cgaagccaag cccagcgcag atgcggtcct caatcgcgcg
     4861 caacgtctcc gcgtcgggct tctctcccgc gcggaagcta accagcaggt gataggtctt
     4921 gtcggcctcg gaacgggtgt tgccgtgctg ggtcgccatc acctcggcca tgacagcggg
     4981 cagggtgttt gcctcgcagt tcgtgacgcg cacgtgaccc aggcgctcgg tcttgccttg
     5041 ctcgtcggtg atgtacttca ccagctccgc gaagtcgctc ttcttgatgg agcgcatggg
     5101 gacgtgcttg gcaatcacgc gcaccccccg gccgttttag cggctaaaaa agtcatggct
     5161 ctgccctcgg gcggaccacg cccatcatga ccttgccaag ctcgtcctgc ttctcttcga
     5221 tcttcgccag cagggcgagg atcgtggcat caccgaaccg cgccgtgcgc gggtcgtcgg
     5281 tgagccagag tttcagcagg ccgcccaggc ggcccaggtc gccattgatg cgggccagct
     5341 cgcggacgtg ctcatagtcc acgacgcccg tgattttgta gccctggccg acggccagca
     5401 ggtaggccga caggctcatg ccggccgccg ccgccttttc ctcaatcgct cttcgttcgt
     5461 ctggaaggca gtacaccttg ataggtgggc tgcccttcct ggttggcttg gtttcatcag
     5521 ccatccgctt gccctcatct gttacgccgg cggtagccgg ccagcctcgc agagcaggat
     5581 tcccgttgag caccgccagg tgcgaataag ggacagtgaa gaaggaacac ccgctcgcgg
     5641 gtgggcctac ttcacctatc ctgcccggct gacgccgttg gatacaccaa ggaaagtcta
     5701 cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat accgaaaaaa
     5761 tcgctataat gaccccgaag cagggttatg cagcggaaaa gcgctgcttc cctgctgttt
     5821 tgtggaatat ctaccgactg gaaacaggca aatgcaggaa attactgaac tgaggggaca
     5881 ggcgagagac gatgccaaag agctacaccg acgagctggc cgagtgggtt gaatcccgcg
     5941 cggccaagaa gcgccggcgt gatgaggctg cggttgcgtt cctggcggtg agggcggatg
     6001 tcgaggcggc gttagcgtcc ggctatgcgc tcgtcaccat ttgggagcac atgcgggaaa
     6061 cggggaaggt caagttctcc tacgagacgt tccgctcgca cgccaggcgg cacatcaagg
     6121 ccaagcccgc cgatgtgccc gcaccgcagg ccaaggctgc ggaacccgcg ccggcaccca
     6181 agacgccgga gccacggcgg ccgaagcagg ggggcaaggc tgaaaagccg gcccccgctg
     6241 cggccccgac cggcttcacc ttcaacccaa caccggacaa aaaggatccc caattctcat
     6301 gtttgacagc ttatcatcga taagctttaa tgcggtagtt tatcacagtt aaattgctaa
     6361 cgcagtcagg caccgtgtat gaaatctaac aatgcgctca tcgtcatcct cggcaccgtc
     6421 accctggatg ctgtaggcat aggcttggtt atgccggtac tgccgggcct cttgcgggat
     6481 atcgtccatt ccgacagcat cgccagtcac tatggcgtgc tgctagcgct atatgcgttg
     6541 atgcaatttc tatgcgcacc cgttctcgga gcactgtccg accgctttgg ccgccgccca
     6601 gtcctgctcg cttcgctact tggagccact atcgactacg cgatcatggc gaccacaccc
     6661 gtcctgtgga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
     6721 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     6781 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     6841 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     6901 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgacc gatgcccttg
     6961 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca
     7021 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc
     7081 attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta
     7141 ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc
     7201 ggcgagaagc aggccattat cgccggcatg gcggccgacg cgctgggcta cgtcttgctg
     7261 gcgttcgcga cgcgaggctg gatggccttc cccattatga ttcttctcgc ttccggcggc
     7321 atcgggatgc ccgcgttgca ggccatgctg tccaggcagg tagatgacga ccatcaggga
     7381 cagcttcaag gatcgctcgc ggctcttacc agcctaactt cgatcattgg accgctgatc
     7441 gtcacggcga tttatgccgc ctcggcgagc acatggaacg ggttggcatg gattgtaggc
     7501 gccgccctat accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc
     7561 tcgacctgaa tggaagccgg cggcacctcg ctaacggatt caccactcca agaattggag
     7621 ccaatcaatt cttgcggaga actgtgaatg cgcaaaccaa cccttggcag aacatatcca
     7681 tcgcgtccgc catctccagc agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc
     7741 cgggaattac gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct
     7801 gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac
     7861 cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat
     7921 attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa
     7981 catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc
     8041 ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg t
//
