LOCUS       sequence-64712-e        6313 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 6313)
  AUTHORS   Marx CJ, Lidstrom ME
  TITLE     Broad-host-range cre-lox system for antibiotic marker recycling in 
            gram-negative bacteria.
  JOURNAL   Biotechniques. 2002 Nov;33(5):1062-7.
  PUBMED    12449384
REFERENCE   2  (bases 1 to 6313)
  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: 
            "Biotechniques."; date: "2002-11"; volume: "33(5)"; pages: "1062-7"
COMMENT     Sequence Label: pCM351
FEATURES             Location/Qualifiers
     source          1..6313
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..861))
                     /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     624..643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(862..966)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    complement(1050..1083)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        join(1103..1108,1109..1125,1126..1131)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             1320..1853
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     protein_bind    complement(1896..1929)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2584..2955)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            2988..3097
                     /note="incP origin of transfer"
     promoter        join(3734..3739,3740..3756,3757..3762)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             3810..5000
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(4162..4181)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(5384..5401)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5555..6143)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5635..5654)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc tgacgtctag atctgaattc agctgtacaa
     1021 ttggtaccat ggatgcatat ggcggccgca taacttcgta tagcatacat tatacgaagt
     1081 tatctacgcg tcaattctcg aattgacata agcctgttcg gttcgtaaac tgtaatgcaa
     1141 gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg
     1201 cgcagtggcg gttttcatgg cttgttatga ctgttttttt gtacagtcta tgcctcgggc
     1261 atccaagcag caagcgcgtt acgccgtggg tcgatgtttg atgttatgga gcagcaacga
     1321 tgttacgcag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaggtg
     1381 gctcaagtat gggcatcatt cgcacatgta ggctcggccc tgaccaagtc aaatccatgc
     1441 gggctgctct tgatcttttc ggtcgtgagt tcggagacgt agccacctac tcccaacatc
     1501 agccggactc cgattacctc gggaacttgc tccgtagtaa gacattcatc gcgcttgctg
     1561 ccttcgacca agaagcggtt gttggcgctc tcgcggctta cgttctgccc aggtttgagc
     1621 agccgcgtag tgagatctat atctatgatc tcgcagtctc cggcgagcac cggaggcagg
     1681 gcattgccac cgcgctcatc aatctcctca agcatgaggc caacgcgctt ggtgcttatg
     1741 tgatctacgt gcaagcagat tacggtgacg atcccgcagt ggctctctat acaaagttgg
     1801 gcatacggga agaagtgatg cactttgata tcgacccaag taccgccacc taacaattcg
     1861 ttcaagccga gatcggcttc ccggccgacg cgtagataac ttcgtatagc atacattata
     1921 cgaagttatg gatccagctt atcgataccg cgggccctac gtacgcgtgt taaccggtga
     1981 gctcactaga ggatccagcc gaccaggctt tccacgcccg cgtgccgctc catgtcgttc
     2041 gcgcggttct cggaaacgcg ctgccgcgtt tcgtgattgt cacgctcaag cccgtagtcc
     2101 cgttcgagcg tcgcgcagag gtcagcgagg gcgcggtagg cccgatacgg ctcatggatg
     2161 gtgtttcggg tcgggtgaat cttgttgatg gcgatatgga tgtgcaggtt gtcggtgtcg
     2221 tgatgcacgg cactgacgcg ctgatgctcg gcgaagccaa gcccagcgca gatgcggtcc
     2281 tcaatcgcgc gcaacgtctc cgcgtcgggc ttctctcccg cgcggaagct aaccagcagg
     2341 tgataggtct tgtcggcctc ggaacgggtg ttgccgtgct gggtcgccat cacctcggcc
     2401 atgacagcgg gcagggtgtt tgcctcgcag ttcgtgacgc gcacgtgacc caggcgctcg
     2461 gtcttgcctt gctcgtcggt gatgtacttc accagctccg cgaagtcgct cttcttgatg
     2521 gagcgcatgg ggacgtgctt ggcaatcacg cgcacccccc ggccgtttta gcggctaaaa
     2581 aagtcatggc tctgccctcg ggcggaccac gcccatcatg accttgccaa gctcgtcctg
     2641 cttctcttcg atcttcgcca gcagggcgag gatcgtggca tcaccgaacc gcgccgtgcg
     2701 cgggtcgtcg gtgagccaga gtttcagcag gccgcccagg cggcccaggt cgccattgat
     2761 gcgggccagc tcgcggacgt gctcatagtc cacgacgccc gtgattttgt agccctggcc
     2821 gacggccagc aggtaggccg acaggctcat gccggccgcc gccgcctttt cctcaatcgc
     2881 tcttcgttcg tctggaaggc agtacacctt gataggtggg ctgcccttcc tggttggctt
     2941 ggtttcatca gccatccgct tgccctcatc tgttacgccg gcggtagccg gccagcctcg
     3001 cagagcagga ttcccgttga gcaccgccag gtgcgaataa gggacagtga agaaggaaca
     3061 cccgctcgcg ggtgggccta cttcacctat cctgcccggc tgacgccgtt ggatacacca
     3121 aggaaagtct acacgaaccc tttggcaaaa tcctgtatat cgtgcgaaaa aggatggata
     3181 taccgaaaaa atcgctataa tgaccccgaa gcagggttat gcagcggaaa agcgctgctt
     3241 ccctgctgtt ttgtggaata tctaccgact ggaaacaggc aaatgcagga aattactgaa
     3301 ctgaggggac aggcgagaga cgatgccaaa gagctacacc gacgagctgg ccgagtgggt
     3361 tgaatcccgc gcggccaaga agcgccggcg tgatgaggct gcggttgcgt tcctggcggt
     3421 gagggcggat gtcgaggcgg cgttagcgtc cggctatgcg ctcgtcacca tttgggagca
     3481 catgcgggaa acggggaagg tcaagttctc ctacgagacg ttccgctcgc acgccaggcg
     3541 gcacatcaag gccaagcccg ccgatgtgcc cgcaccgcag gccaaggctg cggaacccgc
     3601 gccggcaccc aagacgccgg agccacggcg gccgaagcag gggggcaagg ctgaaaagcc
     3661 ggcccccgct gcggccccga ccggcttcac cttcaaccca acaccggaca aaaaggatcc
     3721 ccaattctca tgtttgacag cttatcatcg ataagcttta atgcggtagt ttatcacagt
     3781 taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc
     3841 tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta ctgccgggcc
     3901 tcttgcggga tatcgtccat tccgacagca tcgccagtca ctatggcgtg ctgctagcgc
     3961 tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg agcactgtcc gaccgctttg
     4021 gccgccgccc agtcctgctc gcttcgctac ttggagccac tatcgactac gcgatcatgg
     4081 cgaccacacc cgtcctgtgg atcctctacg ccggacgcat cgtggccggc atcaccggcg
     4141 ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc
     4201 gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg
     4261 ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg
     4321 gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgac
     4381 cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta
     4441 tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag
     4501 cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt
     4561 cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca
     4621 ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggccgac gcgctgggct
     4681 acgtcttgct ggcgttcgcg acgcgaggct ggatggcctt ccccattatg attcttctcg
     4741 cttccggcgg catcgggatg cccgcgttgc aggccatgct gtccaggcag gtagatgacg
     4801 accatcaggg acagcttcaa ggatcgctcg cggctcttac cagcctaact tcgatcactg
     4861 gaccgctgat cgtcacggcg atttatgccg cctcggcgag cacatggaac gggttggcat
     4921 ggattgtagg cgccgcccta taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga
     4981 gccgggccac ctcgacctga atggaagccg gcggcacctc gctaacggat tcaccactcc
     5041 aagaattgga gccaatcaat tcttgcggag aactgtgaat gcgcaaacca acccttggca
     5101 gaacatatcc atcgcgtccg ccatctccag cagccgcacg cggcgcatct cgggcagcgt
     5161 tgggtcctgg ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg
     5221 gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga
     5281 ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt
     5341 ttcgtaaagt ctggaaacgc ggaagtcagc gctcttccgc ttcctcgctc actgactcgc
     5401 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5461 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5521 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5581 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5641 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5701 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5761 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5821 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5881 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5941 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     6001 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6061 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6121 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6181 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6241 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6301 cttggtctga cag
//
