LOCUS       sequence-334743-        5777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Methylase expression vector for 5RM assembly: expresses M.XhoI, 
            M.MunI (MfeI) and M.Ocy1ORF8430P (SpeI) DNA methyltransferases in E.
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5777)
  AUTHORS   Matsumura I
  TITLE     Golden Gate assembly of BioBrick-compliant parts using Type II 
            restriction endonucleases.
  JOURNAL   Biotechniques. 2022 May;72(5):185-193. doi: 10.2144/btn-2021-0083. 
            Epub 2022 Mar 8.
  PUBMED    35255734
REFERENCE   2  (bases 1 to 5777)
  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"; doi: 
            "10.2144/btn-2021-0083"; journalName: "Biotechniques"; date: 
            "2022-05"; volume: "72"; issue: "5"; pages: "185-193"
COMMENT     Sequence Label: Prham-M.XhoI-M.MunI-M.Ocy1ORF8430P-p15A-aadA
FEATURES             Location/Qualifiers
     source          1..5777
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(28..75)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     71..89
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(127..171)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     promoter        331..449
                     /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)"
     RBS             469..477
                     /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)"
     RBS             2212..2220
                     /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)"
     RBS             2935..2943
                     /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      3938..4483
                     /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     4384..4403
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4600..4617
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4751..4842
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4843..5634
                     /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"
ORIGIN
        1 gtgcttctca aatgcctgag gtttcagcaa aaaacccctc aagacccgtt tagaggcccc
       61 aaggggttat gctagttatt gctcagcggt ggcagcagcc taggttaatt aagctgcgct
      121 agtagacgag tccatgtgct ggcgttcaaa tttcgcagca gcggtttctt taccagactc
      181 gacaagctta ctagaggatg tcgtcaacac ggcgaaatag taatcacgag gtcaggttct
      241 taccttaaat tttcgacgga aaaccacgta aaaaacgtcg atttttcaag atacagcgtg
      301 aattttcagg aaatgcggtg agcatcacat caccacaatt cagcaaattg tgaacatcat
      361 cacgttcatc tttccctggt tgccaatggc ccattttcct gtcagtaacg agaaggtcgc
      421 gaattgaggc gctttttaga ctggtcgtaa tgaaattcag ctactagata aggaggtata
      481 tcatgaatct ggatctggca ggcatcccag atgtgttagc ggatgagagc gagagcgcga
      541 aacagttcag cacggcaggc gcagacgaac gtggcgcaat ctttacccgt cgtgaagtgg
      601 tggagtttat cctggactta gttggctata cggaagatcg cgacttagcc cagaccaagc
      661 tgttagaacc gagtgcaggc cacgccgact ttctgttacc gattattggc cgcttagtgc
      721 gcagctatgt tgcgcacggt ggcgatttaa cccgtggtgc acagacctta ggtccggcca
      781 ttcgtgcgta cgaagtgcat gaaagcagtc tggaaacggc gcgtagtgtt gtggttgcgg
      841 agttactgcg tctgggtgtg aaaaaggccg cagccgatgg cctgggccgc acgtggctgg
      901 ttcgcgccga tttcctgatg gcaccattac cacatacgtt cgacttcgtg gtgggcaatc
      961 caccgtatgt tcgtcaagaa ctgattccgg cggtgttatt agcgcgttat cgcgcacgct
     1021 tcaaaaccct gtatgatcgc gcggatctgt atattccgtt ctatgaacgc tgtctggatg
     1081 ttctggcacc gggtggccgt ttaggtttta tctgtacgga tcgttggacc aaaaacaagt
     1141 acggcggtcc attacgcgca atggtgagcg aagagtttag cctgacccat tttgtggacc
     1201 tggtggatac gcaagcgttc ctgagcaatg tgatgaccta cccagccatc accgtgattg
     1261 aacgtccgaa accgaaaagc aaagcgcgtc cgacccgcgt tgcatatcgc ccagcgatca
     1321 gcgcggaagt gtttggcccg ctggcaaaag ccatgacggg caccaaactg aatcataaag
     1381 cgggtgttgt tgaaatgagt ggcgtggtta acggcagcga gccgtggatt ctgcatcagg
     1441 cagatcgctt agcgttagtt cgtcgtttag aggagacgct gccaacgctg gaagaagcgg
     1501 gctgtaaggt tggcatcggc gttgcgaccg gcaatgacgg cgtgtatatc ggcgacatga
     1561 aaacgttaaa cgtggagcca agccgtaagc tgccattagc acgtacccag gacttacgcg
     1621 gtggcagtat tgattggcag ggcaaaggcg ttctgaatcc gttcgaggaa gacggccagg
     1681 ttgtggattt agcgagctat ccaaaatttg cggcgtatct gcaagagcac gcaattcaga
     1741 tcaaagcccg ccatgtggca aagaagaacc cggaacgttg gtttcgtacc attgatcgta
     1801 tttatccagc actggccaag accccgaaac tgctggtgcc ggacattaaa ggcgacgcgc
     1861 atattgtgta tgaagaaggc aaactgtatc cgcatcataa cttatacttt attaccgcga
     1921 atgaatggga tttacgtgcg ctgcaagcag tgttaatgag cggtgtggcg cgtctgtttg
     1981 tgggcaccta tagtaccacc atgcgtggcg gttttctgcg cttccaggcc caatacctgc
     2041 gtcgtattcg tgttccacat tggaaaaatg tgccgaagcc gttacaaaaa gccctgcgcg
     2101 aagcagcggt tgcaggtgat cgtgaggccg ccaatcgtgc aacctatcag ttatatggtt
     2161 taaatgaagc cgagcgtgat attgttgcca cggtttgaaa gctttactag ataaggaggt
     2221 ataccatgga aaataaggtt accgcgtact ccatctataa caaaaaagcg aagaaaaaca
     2281 ccaaagttaa cccgctggac gaagttttcc cgcagctccc gcgtaagaaa taccaggtta
     2341 tctacgcgga cccgccgtgg gactacggtg gtaaaatgca gtacgacaaa tctaccatca
     2401 aatctgaaaa cgaaggtttc aaacgtgaca tcttcatctc ttctgcgtct ttcaaatacc
     2461 cgactctcaa actgaaagaa ctgcaacaac tggacgttcc gtctatcacc gcggacgact
     2521 gcatcctgtt catgtggacc accggtccgc agatggcgaa ctctattctg ctgggtgaat
     2581 cttggggttt cgaatacaaa accgttgcgt tcgtttggga caaaatggtt cacaacccag
     2641 gtcgttacac tctgtctcag accgaatttg ttctggtttt caaaaagggt aagatcccga
     2701 ccccgcgtgg tgctcgcaat gtacgtcagc tgctgcaaat ccaccgtggt cagcactctg
     2761 agaaaccgta cgctgttatc gacggtatca ccaaaatgtt cccggcactg gacaagatcg
     2821 aactgttcgc gcgtaacaac tttgtaggtt gggataactg gggtctggaa atcccggaca
     2881 acaaaatcga gattccgacc cagggtgaaa tcgacgagaa caagtgatac tagataagga
     2941 ggtatatcat gaagaaagcg ccgctcaaca aagttctgat cggtgactgc cgtgaagtta
     3001 tgaaatccct gccggaaaac tacgtttctg cgtgcatcac cgacccaccg tacaactacg
     3061 aatttatcgg tcacaaatgg aacgactctg aaatcaaacg tcgtatcgaa cgtattaaca
     3121 gcaaaaagaa caaaaccctg gttaaaaaca tcccatacgg ttccggtctg gcgggtggtg
     3181 ttcgcaacga gcgttggtat caacgtaatc gtaacaatat cctggactac gagaagtggt
     3241 gcttcgagtg gggcgaagaa ctgttccgta tttgcaagcc gggtgcgact gtactcgtgt
     3301 ttaactctac ccgtaccgtt gctcacgttc aggttgcgct ggaaaacgcg ggtttctatg
     3361 ctcgtgacat cctggtatac aagcgcccgt ctggtatccc gaaaggtgta aacattaaac
     3421 agcagctgga gaaaaaaggt tacgaaaact ctgagaaatg ggacggttgg cacagctgcc
     3481 tgcgtaacga atgggaagcg atctgcgttc tgcaaaaacc gctggtgaac aactatatcg
     3541 aaactctcct ggaatacggt actggtctct tctacaccaa agaatacctg ggcggcttcc
     3601 aatctaacat tctggacggt atccagcgtg aacgtaccga agacttcaac gtgcactgca
     3661 ccgttaaacc gatcgcgctg atgcgtaaac tggttgaaat cttcgttccg cgtctggaag
     3721 actctatcct gatcgacccg ttcgctggtt ctggtactac cctgctggct gcgaaagagc
     3781 tgggtatctc ttacatcggt atcgagatcg tttctaacta catcgaaatc atcaacaagc
     3841 gtctcgacaa cttctctggt ctgcaaggca tcatcccgtt cttccaatga tactagtacg
     3901 cctagaaata ttttatctga ttaataagat gatcttcttg agatcgtttt ggtctgcgcg
     3961 taatctcttg ctctgaaaac gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct
     4021 gagctaccaa ctctttgaac cgaggtaact ggcttggagg agcgcagtca ccaaaacttg
     4081 tcctttcagt ttagccttaa ccggcgcatg acttcaagac taactcctct aaatcaatta
     4141 ccagtggctg ctgccagtgg tgcttttgca tgtctttccg ggttggactc aagacgatag
     4201 ttaccggata aggcgcagcg gtcggactga acggggggtt cgtgcataca gtccagcttg
     4261 gagcgaactg cctacccgga actgagtgtc aggcgtggaa tgagacaaac gcggccataa
     4321 cagcggaatg acaccggtaa accgaaaggc aggaacagga gagcgcacga gggagccgcc
     4381 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccaccact gatttgagcg
     4441 tcagatttcg tgatgcttgt caggggggcg gagcctatgg aaaaacggct ttgccgcggc
     4501 cctctcactt ccctgttaag tatcttcctg gcatcttcca ggaaatctcc gccccgttcg
     4561 taagccattt ccgctcgccg cagtcgaacg accgagcgta gcgagtcagt gagcgaggaa
     4621 gcggaatata tcctgtatca catattctgc tgacgcaccg gtgcagcctt ttttctcctg
     4681 ccacatgaag cacttcactg acaccctcat cagtgccaac atagtaagcc agtatacact
     4741 ccgctagcta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4801 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgaggga agcggtgatc
     4861 gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg
     4921 acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt
     4981 gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg
     5041 atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta
     5101 gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa
     5161 ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg
     5221 atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta
     5281 ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta
     5341 aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta
     5401 gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat
     5461 gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa
     5521 gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg
     5581 gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataatgtcta
     5641 acaattcgtt caagccgagg ggccgcaaga tccggccacg atgacccggt cgtcggttca
     5701 gggcagggtc gttaaatagc cgcttatgtc tattgctggt ttaccggttt attgactacc
     5761 ggaagcagtg tgaccgt
//
