LOCUS       sequence-334750-        6437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Methylase expression vector for 5RM assembly: expresses M.XbaI, 
            M.EcoRI and M.SalI DNA methyltransferases in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6437)
  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 6437)
  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.XbaI-M.EcoRI-M.SalI-p15A-aadA
FEATURES             Location/Qualifiers
     source          1..6437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      669..1214
                     /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     1115..1134
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1331..1348
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1482..1573
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1574..2365
                     /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(2536..2583)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2579..2597
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2635..2679)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     promoter        2839..2957
                     /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             4282..4290
                     /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             5284..5292
                     /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)"
ORIGIN
        1 gcgtatctga gtacggccga cgaaaagatt cgtggcggtt atttagtgag taaacgtagt
       61 ccgtggtata aacaggaaca acgcgagcca gcaccgattt tactgaccta tatgggccgt
      121 ggcaaggatg accagcatcc attacgtttt atccgtaatg atagtgatgc cgttgcgacg
      181 aatatgtact taatgttata tccgacggca ttactgcaac gttacttagc cggcgaccca
      241 gaacgcatca agcaggtgca caaagcgtta ttagcgatta ccgcagccga tctgcgcggt
      301 ggtggtcgtg tttacggcgg tggcctgcac aagatggaac cgaaggagtt agccgcatta
      361 ccggccgatg gcattgcgac cctggaccca gttttacgtg aagatattag tatggttagt
      421 gttccgccgc gtaagcgcac gggccgtccg cagatgccag gtccgagtgc aagtgaagtg
      481 cgcgcctggg cacgcgcgaa tggtgtgtgc gttccagatc gtggccgttt acgtccggaa
      541 gtttgggacg cctggcgtca ggcgcacgcg ggtgaggcaa gcccattaaa catcgacgcc
      601 ggtgatcagg tggccctgtg gtgaaagctt tactagaaat attttatctg attaataaga
      661 tgatcttctt gagatcgttt tggtctgcgc gtaatctctt gctctgaaaa cgaaaaaacc
      721 gccttgcagg gcggtttttc gaaggttctc tgagctacca actctttgaa ccgaggtaac
      781 tggcttggag gagcgcagtc accaaaactt gtcctttcag tttagcctta accggcgcat
      841 gacttcaaga ctaactcctc taaatcaatt accagtggct gctgccagtg gtgcttttgc
      901 atgtctttcc gggttggact caagacgata gttaccggat aaggcgcagc ggtcggactg
      961 aacggggggt tcgtgcatac agtccagctt ggagcgaact gcctacccgg aactgagtgt
     1021 caggcgtgga atgagacaaa cgcggccata acagcggaat gacaccggta aaccgaaagg
     1081 caggaacagg agagcgcacg agggagccgc cagggggaaa cgcctggtat ctttatagtc
     1141 ctgtcgggtt tcgccaccac tgatttgagc gtcagatttc gtgatgcttg tcaggggggc
     1201 ggagcctatg gaaaaacggc tttgccgcgg ccctctcact tccctgttaa gtatcttcct
     1261 ggcatcttcc aggaaatctc cgccccgttc gtaagccatt tccgctcgcc gcagtcgaac
     1321 gaccgagcgt agcgagtcag tgagcgagga agcggaatat atcctgtatc acatattctg
     1381 ctgacgcacc ggtgcagcct tttttctcct gccacatgaa gcacttcact gacaccctca
     1441 tcagtgccaa catagtaagc cagtatacac tccgctagct atttgtttat ttttctaaat
     1501 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1561 aaaaaggaag agtatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt
     1621 agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc
     1681 cgcagtggat ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt
     1741 aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc
     1801 ccctggagag agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat
     1861 cattccgtgg cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga
     1921 cattcttgca ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac
     1981 aaaagcaaga gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc
     2041 ggttcctgaa caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc
     2101 gcccgactgg gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag
     2161 cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct
     2221 gccggcccag tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga
     2281 agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga
     2341 gatcaccaag gtagtcggca aataatgtct aacaattcgt tcaagccgag gggccgcaag
     2401 atccggccac gatgacccgg tcgtcggttc agggcagggt cgttaaatag ccgcttatgt
     2461 ctattgctgg tttaccggtt tattgactac cggaagcagt gtgaccgtgt gcttctcaaa
     2521 tgcctgaggt ttcagcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc
     2581 tagttattgc tcagcggtgg cagcagccta ggttaattaa gctgcgctag tagacgagtc
     2641 catgtgctgg cgttcaaatt tcgcagcagc ggtttcttta ccagactcga caagcttact
     2701 agaggatgtc gtcaacacgg cgaaatagta atcacgaggt caggttctta ccttaaattt
     2761 tcgacggaaa accacgtaaa aaacgtcgat ttttcaagat acagcgtgaa ttttcaggaa
     2821 atgcggtgag catcacatca ccacaattca gcaaattgtg aacatcatca cgttcatctt
     2881 tccctggttg ccaatggccc attttcctgt cagtaacgag aaggtcgcga attgaggcgc
     2941 tttttagact ggtcgtaatg aaattcagct actagagcat gcgtacttag aggaaataaa
     3001 ccatgaaaaa tgcgaccacc caggctcgtg cggtgcaggg tgactgtctg gttgaactcg
     3061 cgcgtctgga agaatgcgaa gctgatctcg tttacctgga cccgccgttc ttcaccaacc
     3121 gtcgtcactc ctccattagc cgtgaccgta ttaatgcttt ctctttcgac gacgcgtggg
     3181 gtgatctggg tgagtatgca gattttatgg aacaacgtct ccgtgaggcg caccgtgtgc
     3241 tgaaacattc tggttctatc ttcgttcact gcgatacttc tgcgaacttt ctcctgcgta
     3301 ctctcctcaa caacatcttc ggcgcgtccc agtttcgcag cgaaatcatc tggtcctaca
     3361 agcgctggtc taactctgct cgtaacctgc tgccagcgca tcagaccatc ctgttctact
     3421 ctaaatctga cgcgtacaaa ttcaatgttc tccacggttc ttattctgaa accactaatg
     3481 tagatcagat cctccagctg cgccaacgtg acgctgacgg tgttagcaaa tatgcgactg
     3541 acaaactcgg taacaccatc tatggcaccg aaaagaatgg cgtgccgctg aacgacgttt
     3601 gggcaatccc gtttctcaat ccgaaagcga aagaacgtac cggctatccg actcagaaac
     3661 caattctgct gctggaacgc atcatcgaaa tctccaccga tccaggtgac ttcatcgttg
     3721 acccgttctg tggttctggt actaccctgg tggcggcagc gatcctgggc cgtcgtgcat
     3781 tcggtatcga cacttctcgc gaagcggttg ctctggcaaa ccgccgtatc gaggaaccgg
     3841 tgcgtaccga ttctgcactc ctgaaaaagg gtcgtctggc atacgcgact gctaacctca
     3901 aagcactcgc ggttctcgaa ggtctggact tcgtgccagt acaccgtaac agcggtattg
     3961 atgcttttct gcgctctcca gaaggtgaac cgctggttcc agtacgtgtg caacgtaacg
     4021 gtgaatctat cagcgacgcg gctgcttccc tggcgcgtgc tgctattagc aagaaggcga
     4081 aaaacgcaat cctcgttcgt actaatgaag agactggtct gtttagcacc gacgcactcc
     4141 caccgtttat gcacctgatt gagtctaccg cactgcgcat taaatctagc ctggatgcgt
     4201 ccctcatcgc gggtgagact aaatccgcag caaacaactc tttcaaacca aaaccgctcc
     4261 gtggctccgc gtgatactag ataaggaggt ataccatggc gcgtaacgcg accaataaac
     4321 tgctgcataa agcgaaaaaa tctaaatctg acgagttcta cacccaatac tgtgacattg
     4381 agaacgaact ccagtactac cgtgaacact tctctgacaa agttgtttac tgcaactgcg
     4441 atgacccgcg tgtttctaac ttcttcaaat acttcgctgt aaatttcgac aacctgggtc
     4501 tgaagaaact gatcgcgtct tgctatgtgg aaaacaaaga aggtttctct tcttctgaag
     4561 cggctaaaaa cggtttctac tacgaatacc acaaggagaa cggcaaaaaa ctcgttttcg
     4621 atgacatttc tgtgtcttct ttttgcggtg acggtgactt ccgttctagc gaatctatcg
     4681 acctgctcaa gaaatccgac atcgttgtta ccaacccgcc gttttctctc ttccgtgaat
     4741 atctggacca actcattaag tacgacaaaa agtttctgat catcgcgaac gttaacagca
     4801 tcacctataa ggaagttttc aacctgatca aagagaacaa aatctggctg ggcgtacatc
     4861 tgggtcgtgg cgtttccggt ttcatcgttc cggagcacta tgaactgtat ggcaccgaag
     4921 ctcgtatcga ctctaatggt aatcgtatca tttctccgaa caactgcctg tggctgacca
     4981 acctcgacgt tttcatccgt cataaggacc tgccgctcac tcgtaaatac tttggtaatg
     5041 aaagctctta cccgaaatat gataattacg atgcgatcaa tgtgaataaa accaaagaca
     5101 tcccgctgga ctacaacggt gttatgggtg ttccgatcac ctttctgcac aaattcaacc
     5161 cggaacagtt cgagctgatt aagttccgca aaggtgttga cgagaaagac ctgtctatca
     5221 atggcaaatg cccgtatttt cgtattctca tcaagaataa acgcctccaa aaatgatact
     5281 agataaggag gtatagcatg cacagcgaag cccgtgaggc ggaagcacgt cgcttactgc
     5341 tgcaagaaac cttagacgcg gagcgtacct tcttagaacg taaccagtgg ggtcaatttg
     5401 caaccccacc gagcttagca ggcgagatta tgcgttatac gattgatctg catgaagaga
     5461 cgcgtattaa ctttttagaa ccgagttgtg gcagtggtag tttctttagc gcactgttac
     5521 gcaacttagg cgataagaaa atcgagcacg cagtgggcgt tgaattagat ccacgcttta
     5581 gcaaagcggc gagcgatctg tggaccggcc aaggcctgcg tgttattgag ggtgatttca
     5641 cgagtccaag cctggttagc ggtccggttg ccagcctgct ggtggcgaat ccgccatacg
     5701 ttcgtcacca ccatctgggt attgatcaga aacgtgatct ggttgcacgt tgcgccgatc
     5761 agttaggtat taagccgagt ggtttaagcg gtctgtatct gtatttcgtt ctgctgagcc
     5821 atcgtttatt acgtgcggat gcggttagta cgtggctgat tccaagcgaa tttatggacg
     5881 ttaactatgg tacggcgctg aaagaatatc tggccacccg tgtgcagtta gtgcgcattc
     5941 accagtatga tgcagcggag gttcagttcg atgacgccct ggtgaccagt agtgtggttg
     6001 tgtttcgtaa tagcccacca cgcccaggtc acaccgcaga atttagtttc ggcggtacgt
     6061 taagtgagcc gaaggtgacc catcagatcc cgagcgcagc cttaacgcca gaagcgaaat
     6121 ggagccgtta tgtgaccggt gttatgccag cagacattaa cctgaaacag acgggtccaa
     6181 agctgagcga ctttttcaaa atccgtcgtg gcttagcgac cggcagcaat gcgtttttca
     6241 tcattccacg tagtgaggcc gaacgtctgg gcattaagcg taattttctg cgcccgattc
     6301 tgccgagccc acgcaaactg aaaggcgatg cgatcacggc ggacgcgagt ggctggccgg
     6361 acattccgga gcagctggcg ctgctggact gcccgctgcc gatcgaggac ctgttactgg
     6421 aaaatccagc attagca
//
