LOCUS       sequence-64710-e        6760 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 6760)
  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 6760)
  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: pCM184
FEATURES             Location/Qualifiers
     source          1..6760
                     /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)."
     primer_bind     complement(1206..1225)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1217..2032
                     /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             1217..2032
                     /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(1290..1309)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     protein_bind    complement(2343..2376)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(3031..3402)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            3435..3544
                     /note="incP origin of transfer"
     promoter        join(4181..4186,4187..4203,4204..4209)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             4257..5447
                     /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(4609..4628)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(5831..5848)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6002..6590)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6082..6101)
                     /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 tatctagacc tgcagggggg ggggggaaag ccacgttgtg tctcaaaatc tctgatgtta
     1141 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag
     1201 taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcga ggccgcgatt
     1261 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     1321 atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa
     1381 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     1441 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
     1501 gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga
     1561 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     1621 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     1681 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     1741 tgaacaagtc tggaaagaaa tgcataagct tttgccattc tcaccggatt cagtcgtcac
     1801 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     1861 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     1921 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     1981 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt
     2041 ggttaattgg ttgtaacact ggcagagcat tacgctgact tgacgggacg gcggctttgt
     2101 tgaataaatc gaacttttgc tgagttgaag gatcagatca cgcatcttcc cgacaacgca
     2161 gaccgttccg tggcaaagca aaagttcaaa atcaccaact ggtccaccta caacaaagct
     2221 ctcatcaacc gtggctccct cactttctgg ctggatgatg gggcgattca ggcctggtat
     2281 gagtcagcaa caccttcttc acgaggcaga cctcagcgcc cccccccccc tgcaggtcct
     2341 agataacttc gtatagcata cattatacga agttatggat ccagcttatc gataccgcgg
     2401 gccctacgta cgcgtgttaa ccggtgagct cactagagga tccagccgac caggctttcc
     2461 acgcccgcgt gccgctccat gtcgttcgcg cggttctcgg aaacgcgctg ccgcgtttcg
     2521 tgattgtcac gctcaagccc gtagtcccgt tcgagcgtcg cgcagaggtc agcgagggcg
     2581 cggtaggccc gatacggctc atggatggtg tttcgggtcg ggtgaatctt gttgatggcg
     2641 atatggatgt gcaggttgtc ggtgtcgtga tgcacggcac tgacgcgctg atgctcggcg
     2701 aagccaagcc cagcgcagat gcggtcctca atcgcgcgca acgtctccgc gtcgggcttc
     2761 tctcccgcgc ggaagctaac cagcaggtga taggtcttgt cggcctcgga acgggtgttg
     2821 ccgtgctggg tcgccatcac ctcggccatg acagcgggca gggtgtttgc ctcgcagttc
     2881 gtgacgcgca cgtgacccag gcgctcggtc ttgccttgct cgtcggtgat gtacttcacc
     2941 agctccgcga agtcgctctt cttgatggag cgcatgggga cgtgcttggc aatcacgcgc
     3001 accccccggc cgttttagcg gctaaaaaag tcatggctct gccctcgggc ggaccacgcc
     3061 catcatgacc ttgccaagct cgtcctgctt ctcttcgatc ttcgccagca gggcgaggat
     3121 cgtggcatca ccgaaccgcg ccgtgcgcgg gtcgtcggtg agccagagtt tcagcaggcc
     3181 gcccaggcgg cccaggtcgc cattgatgcg ggccagctcg cggacgtgct catagtccac
     3241 gacgcccgtg attttgtagc cctggccgac ggccagcagg taggccgaca ggctcatgcc
     3301 ggccgccgcc gccttttcct caatcgctct tcgttcgtct ggaaggcagt acaccttgat
     3361 aggtgggctg cccttcctgg ttggcttggt ttcatcagcc atccgcttgc cctcatctgt
     3421 tacgccggcg gtagccggcc agcctcgcag agcaggattc ccgttgagca ccgccaggtg
     3481 cgaataaggg acagtgaaga aggaacaccc gctcgcgggt gggcctactt cacctatcct
     3541 gcccggctga cgccgttgga tacaccaagg aaagtctaca cgaacccttt ggcaaaatcc
     3601 tgtatatcgt gcgaaaaagg atggatatac cgaaaaaatc gctataatga ccccgaagca
     3661 gggttatgca gcggaaaagc gctgcttccc tgctgttttg tggaatatct accgactgga
     3721 aacaggcaaa tgcaggaaat tactgaactg aggggacagg cgagagacga tgccaaagag
     3781 ctacaccgac gagctggccg agtgggttga atcccgcgcg gccaagaagc gccggcgtga
     3841 tgaggctgcg gttgcgttcc tggcggtgag ggcggatgtc gaggcggcgt tagcgtccgg
     3901 ctatgcgctc gtcaccattt gggagcacat gcgggaaacg gggaaggtca agttctccta
     3961 cgagacgttc cgctcgcacg ccaggcggca catcaaggcc aagcccgccg atgtgcccgc
     4021 accgcaggcc aaggctgcgg aacccgcgcc ggcacccaag acgccggagc cacggcggcc
     4081 gaagcagggg ggcaaggctg aaaagccggc ccccgctgcg gccccgaccg gcttcacctt
     4141 caacccaaca ccggacaaaa aggatcccca attctcatgt ttgacagctt atcatcgata
     4201 agctttaatg cggtagttta tcacagttaa attgctaacg cagtcaggca ccgtgtatga
     4261 aatctaacaa tgcgctcatc gtcatcctcg gcaccgtcac cctggatgct gtaggcatag
     4321 gcttggttat gccggtactg ccgggcctct tgcgggatat cgtccattcc gacagcatcg
     4381 ccagtcacta tggcgtgctg ctagcgctat atgcgttgat gcaatttcta tgcgcacccg
     4441 ttctcggagc actgtccgac cgctttggcc gccgcccagt cctgctcgct tcgctacttg
     4501 gagccactat cgactacgcg atcatggcga ccacacccgt cctgtggatc ctctacgccg
     4561 gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg
     4621 acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg
     4681 tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac
     4741 cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc
     4801 aggagtcgca taagggagag cgtcgaccga tgcccttgag agccttcaac ccagtcagct
     4861 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca
     4921 tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc
     4981 gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg
     5041 ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg
     5101 ccggcatggc ggccgacgcg ctgggctacg tcttgctggc gttcgcgacg cgaggctgga
     5161 tggccttccc cattatgatt cttctcgctt ccggcggcat cgggatgccc gcgttgcagg
     5221 ccatgctgtc caggcaggta gatgacgacc atcagggaca gcttcaagga tcgctcgcgg
     5281 ctcttaccag cctaacttcg atcactggac cgctgatcgt cacggcgatt tatgccgcct
     5341 cggcgagcac atggaacggg ttggcatgga ttgtaggcgc cgccctatac cttgtctgcc
     5401 tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg gaagccggcg
     5461 gcacctcgct aacggattca ccactccaag aattggagcc aatcaattct tgcggagaac
     5521 tgtgaatgcg caaaccaacc cttggcagaa catatccatc gcgtccgcca tctccagcag
     5581 ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca tgatcgtgct
     5641 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc
     5701 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac
     5761 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgct
     5821 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5881 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5941 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     6001 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     6061 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     6121 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6181 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6241 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6301 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6361 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6421 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     6481 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6541 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6601 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6661 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6721 aatcaatcta aagtatatat gagtaaactt ggtctgacag
//
