LOCUS       sequence-300479-        3353 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses M.EcoRI DNA methyltransferase in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3353)
  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 3353)
  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.EcoRI-p15A-aadA
FEATURES             Location/Qualifiers
     source          1..3353
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1021..1566
                     /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     1467..1486
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1683..1700
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1834..1925
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1926..2717
                     /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(2888..2935)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2931..2949
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2987..3031)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     promoter        3191..3309
                     /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 catggcgcgt aacgcgacca ataaactgct gcataaagcg aaaaaatcta aatctgacga
       61 gttctacacc caatactgtg acattgagaa cgaactccag tactaccgtg aacacttctc
      121 tgacaaagtt gtttactgca actgcgatga cccgcgtgtt tctaacttct tcaaatactt
      181 cgctgtaaat ttcgacaacc tgggtctgaa gaaactgatc gcgtcttgct atgtggaaaa
      241 caaagaaggt ttctcttctt ctgaagcggc taaaaacggt ttctactacg aataccacaa
      301 ggagaacggc aaaaaactcg ttttcgatga catttctgtg tcttcttttt gcggtgacgg
      361 tgacttccgt tctagcgaat ctatcgacct gctcaagaaa tccgacatcg ttgttaccaa
      421 cccgccgttt tctctcttcc gtgaatatct ggaccaactc attaagtacg acaaaaagtt
      481 tctgatcatc gcgaacgtta acagcatcac ctataaggaa gttttcaacc tgatcaaaga
      541 gaacaaaatc tggctgggcg tacatctggg tcgtggcgtt tccggtttca tcgttccgga
      601 gcactatgaa ctgtatggca ccgaagctcg tatcgactct aatggtaatc gtatcatttc
      661 tccgaacaac tgcctgtggc tgaccaacct cgacgttttc atccgtcata aggacctgcc
      721 gctcactcgt aaatactttg gtaatgaaag ctcttacccg aaatatgata attacgatgc
      781 gatcaatgtg aataaaacca aagacatccc gctggactac aacggtgtta tgggtgttcc
      841 gatcaccttt ctgcacaaat tcaacccgga acagttcgag ctgattaagt tccgcaaagg
      901 tgttgacgag aaagacctgt ctatcaatgg caaatgcccg tattttcgta ttctcatcaa
      961 gaataaacgc ctccaaaaat gatactagaa atattttatc tgattaataa gatgatcttc
     1021 ttgagatcgt tttggtctgc gcgtaatctc ttgctctgaa aacgaaaaaa ccgccttgca
     1081 gggcggtttt tcgaaggttc tctgagctac caactctttg aaccgaggta actggcttgg
     1141 aggagcgcag tcaccaaaac ttgtcctttc agtttagcct taaccggcgc atgacttcaa
     1201 gactaactcc tctaaatcaa ttaccagtgg ctgctgccag tggtgctttt gcatgtcttt
     1261 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcggac tgaacggggg
     1321 gttcgtgcat acagtccagc ttggagcgaa ctgcctaccc ggaactgagt gtcaggcgtg
     1381 gaatgagaca aacgcggcca taacagcgga atgacaccgg taaaccgaaa ggcaggaaca
     1441 ggagagcgca cgagggagcc gccaggggga aacgcctggt atctttatag tcctgtcggg
     1501 tttcgccacc actgatttga gcgtcagatt tcgtgatgct tgtcaggggg gcggagccta
     1561 tggaaaaacg gctttgccgc ggccctctca cttccctgtt aagtatcttc ctggcatctt
     1621 ccaggaaatc tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga acgaccgagc
     1681 gtagcgagtc agtgagcgag gaagcggaat atatcctgta tcacatattc tgctgacgca
     1741 ccggtgcagc cttttttctc ctgccacatg aagcacttca ctgacaccct catcagtgcc
     1801 aacatagtaa gccagtatac actccgctag ctatttgttt atttttctaa atacattcaa
     1861 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     1921 agagtatgag ggaagcggtg atcgccgaag tatcgactca actatcagag gtagttggcg
     1981 tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg
     2041 atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc gtaaggcttg
     2101 atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct tcccctggag
     2161 agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac atcattccgt
     2221 ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat gacattcttg
     2281 caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg acaaaagcaa
     2341 gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat ccggttcctg
     2401 aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg ccgcccgact
     2461 gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac agcgcagtaa
     2521 ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc ctgccggccc
     2581 agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa gaagatcgct
     2641 tggcctcgcg cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc gagatcacca
     2701 aggtagtcgg caaataatgt ctaacaattc gttcaagccg aggggccgca agatccggcc
     2761 acgatgaccc ggtcgtcggt tcagggcagg gtcgttaaat agccgcttat gtctattgct
     2821 ggtttaccgg tttattgact accggaagca gtgtgaccgt gtgcttctca aatgcctgag
     2881 gtttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat gctagttatt
     2941 gctcagcggt ggcagcagcc taggttaatt aagctgcgct agtagacgag tccatgtgct
     3001 ggcgttcaaa tttcgcagca gcggtttctt taccagactc gacaagctta ctagaggatg
     3061 tcgtcaacac ggcgaaatag taatcacgag gtcaggttct taccttaaat tttcgacgga
     3121 aaaccacgta aaaaacgtcg atttttcaag atacagcgtg aattttcagg aaatgcggtg
     3181 agcatcacat caccacaatt cagcaaattg tgaacatcat cacgttcatc tttccctggt
     3241 tgccaatggc ccattttcct gtcagtaacg agaaggtcgc gaattgaggc gctttttaga
     3301 ctggtcgtaa tgaaattcag ctactagagc atgcgtactt agaggaaata aac
//
