LOCUS       sequence-300481-        3248 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses M.AvaIII DNA methyltransferase (M.NsiI ortholog) in E. 
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3248)
  AUTHORS   Matsumura I
  TITLE     Methylase-assisted subcloning for high throughput BioBrick assembly.
  JOURNAL   PeerJ. 2020 Sep 11;8:e9841. doi: 10.7717/peerj.9841. eCollection 
            2020.
  PUBMED    32974095
REFERENCE   2  (bases 1 to 3248)
  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: "PeerJ."; 
            date: "2020-09-11"; volume: "8:e9841. doi"; pages: " 
            10.7717/peerj.9841. eCollection 2020"
COMMENT     Sequence Label: Prham-M.AvaIII-p15A-aadA
FEATURES             Location/Qualifiers
     source          1..3248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             6..14
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     rep_origin      946..1491
                     /direction=RIGHT
                     /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     1392..1411
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1608..1625
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1759..1850
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1851..2642
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     terminator      complement(2813..2860)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2856..2874
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2912..2956)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     promoter        3116..3234
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
ORIGIN
        1 ctagataagg aggtataaca tgtttgaaac ctacgaagac gaagcgcata tcctgttcca
       61 cggtgacgcg ctgaacatcc tctcttctaa catcccgtct gaatctgttg acctgatctt
      121 catcgacccg ccatacaaca tcggtaaaca cttttccaac ttccacgaca aatgggagtc
      181 tgaagaggaa tacgcgaact gggcgtacaa gtggctggac gagtgcattc gtgtgctcaa
      241 gtctcacggt actatttacg ttatgacctc tactcaagcg atgccgtact tcgacatcta
      301 cctgcgtcaa aaactgacca tcctgagccg tatcgtttgg cattacgact cttctggtgt
      361 tcaggcgacc aaatacttcg gttctatgta cgaaccgatc ctgcattgcg tgaagaacaa
      421 gtctaactac atcttcaaca gcgaagaaat caagatcgaa gcgaaaaccg gtgcgcagcg
      481 taaactgatc gactaccgta aagcggttcc ggctccgtac aacaccgaaa aagttccggg
      541 taatgtgtgg tacttcagcc gtgtacgtta ccgtatgccg gaatatgaga accacccgtc
      601 ccagaaaccg gaatctctga ttgagcgtat catcctgtcc tctagcaacg aggatagcct
      661 ggttctggac ccgttcgcgg gtactttcac cgttgcgtct gttgcgaaac gtctgggtcg
      721 caaaagcatc tctatcgaat ctcaggaaga gtacctgaaa atcggtctgc gtcgtgttct
      781 cggttgggag gagtacaaag gtgaacaact gtctccgccg atcaaaaccc acatctctcg
      841 taacaaatct caccagtctc tggattctct ccagcaaagc ctctttaatg cggacaccac
      901 cgcgtgatac tagaaatatt ttatctgatt aataagatga tcttcttgag atcgttttgg
      961 tctgcgcgta atctcttgct ctgaaaacga aaaaaccgcc ttgcagggcg gtttttcgaa
     1021 ggttctctga gctaccaact ctttgaaccg aggtaactgg cttggaggag cgcagtcacc
     1081 aaaacttgtc ctttcagttt agccttaacc ggcgcatgac ttcaagacta actcctctaa
     1141 atcaattacc agtggctgct gccagtggtg cttttgcatg tctttccggg ttggactcaa
     1201 gacgatagtt accggataag gcgcagcggt cggactgaac ggggggttcg tgcatacagt
     1261 ccagcttgga gcgaactgcc tacccggaac tgagtgtcag gcgtggaatg agacaaacgc
     1321 ggccataaca gcggaatgac accggtaaac cgaaaggcag gaacaggaga gcgcacgagg
     1381 gagccgccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccaccactga
     1441 tttgagcgtc agatttcgtg atgcttgtca ggggggcgga gcctatggaa aaacggcttt
     1501 gccgcggccc tctcacttcc ctgttaagta tcttcctggc atcttccagg aaatctccgc
     1561 cccgttcgta agccatttcc gctcgccgca gtcgaacgac cgagcgtagc gagtcagtga
     1621 gcgaggaagc ggaatatatc ctgtatcaca tattctgctg acgcaccggt gcagcctttt
     1681 ttctcctgcc acatgaagca cttcactgac accctcatca gtgccaacat agtaagccag
     1741 tatacactcc gctagctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1801 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagggaag
     1861 cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc
     1921 tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc
     1981 cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc
     2041 gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc
     2101 gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta
     2161 agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc
     2221 cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg
     2281 ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg
     2341 aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct ggcgatgagc
     2401 gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc
     2461 cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca
     2521 tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag
     2581 atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta gtcggcaaat
     2641 aatgtctaac aattcgttca agccgagggg ccgcaagatc cggccacgat gacccggtcg
     2701 tcggttcagg gcagggtcgt taaatagccg cttatgtcta ttgctggttt accggtttat
     2761 tgactaccgg aagcagtgtg accgtgtgct tctcaaatgc ctgaggtttc agcaaaaaac
     2821 ccctcaagac ccgtttagag gccccaaggg gttatgctag ttattgctca gcggtggcag
     2881 cagcctaggt taattaagct gcgctagtag acgagtccat gtgctggcgt tcaaatttcg
     2941 cagcagcggt ttctttacca gactcgacaa gcttactaga ggatgtcgtc aacacggcga
     3001 aatagtaatc acgaggtcag gttcttacct taaattttcg acggaaaacc acgtaaaaaa
     3061 cgtcgatttt tcaagataca gcgtgaattt tcaggaaatg cggtgagcat cacatcacca
     3121 caattcagca aattgtgaac atcatcacgt tcatctttcc ctggttgcca atggcccatt
     3181 ttcctgtcag taacgagaag gtcgcgaatt gaggcgcttt ttagactggt cgtaatgaaa
     3241 ttcagcta
//
