LOCUS       sequence-437782-        7171 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BsaI-associated switch methylase subunit M.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7171)
  AUTHORS   Flores-Fernandez CN, Lin D, Robins K, O'Callaghan CA
  TITLE     DNA methylases for site-selective inhibition of type IIS restriction
            enzyme activity.
  JOURNAL   Appl Microbiol Biotechnol. 2024 Jan 25;108(1):174. doi: 
            10.1007/s00253-024-13015-7.
  PUBMED    38270650
REFERENCE   2  (bases 1 to 7171)
  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: "Appl 
            Microbiol Biotechnol."; date: "2024-01-25"; volume: "108(1):174. 
            doi"; pages: " 10.1007/s00253-024-13015-7"
COMMENT     Sequence Label: POC1475
FEATURES             Location/Qualifiers
     source          1..7171
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..32,33..38)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(213..260)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     256..274
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1803..1820)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1830..1847)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1866..1888
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1903..1927
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     complement(1927..1946)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1928..1946)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2030..2049)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2255..2332
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(2352..2371)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4726..4917
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4911..4933)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5019..5158
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5074..5093
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5173..5190)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5344..5932)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5424..5443)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6103..6894,6895..6963))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6726..6745
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6964..7068)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7136..7154
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
      241 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
      301 cgggctttgt tagcagccgg atcctcgagt tagttcgcct ctttaccact caacgcagtt
      361 cctaagatgt cacgaagctg atggcgaaga gtttggatgg cctcttgctg tttggcgtat
      421 tgtgctaaca gctcgtctgg gtcatgggat acggtttctg cctggtgggg gttcttaata
      481 tcaagattga agttacgggc gataacttca tcaatagaga ctttccaggc ctgctcattc
      541 tcgacgcgac ttgcaaaccc atccgcttcg ttaccccacc aatcaatttc tgcctgaaac
      601 tcctcaaatt tcattggttt cgtcttgctg tagtttttaa cacctgcggg gtaagggtgt
      661 tcataaaacc agatctcctt ggtcggctgc cctttcgtga aaaataacag gttagttttg
      721 atccccgtat aggggttaaa aacgccgtta ggtaaacgga caattgtgtg caaattgcac
      781 tcttcggtca gcaacttttt aatcttagtt ttaacacctt caccaaaaag cgtaccatcg
      841 ggcaacacca cggcggcacg accgttctta gctaagacct caataataag ctgaagaaat
      901 aagtccgccg tctcacgtgt ctgcatgtca gaggggaagt tcttttcaat gccatcttcc
      961 tcagtacccc cgaatggggg gttagtgatg ataacatcca tttgctcgtc ccacgatgaa
     1021 agtggtttat taagagtatt gtcgtggcgg atttgaacgg ggacttcgat gccgtgaagc
     1081 agcatgttgg tcgtacacaa caagtgtggt aattgcttct tttcgacccc atgaatttgt
     1141 ttctgcaaga tttgatggtc agtcacagta tgggcataat ggtttttaac gtggtcgaag
     1201 gcgcatgcca aaaatccccc cgtaccacaa gccggatcca tgatcgactc acccagttta
     1261 gggtctacac gttcaaccat gaagcgagta acagcgcgag gcgtataaaa ttcaccagca
     1321 ttaccggcag cctgcaagtc acgcaaaatt tgctcgtaga tatctccaaa aagatggcgc
     1381 tctgaagcac gtgtaaagtc aatctcattt aacttgttga taacttggcg cagcaacgtc
     1441 ccgtttttca tgtagttata agcgtcggag aaagcctgct ttactacata cccgcggggg
     1501 ttgatgtcga tctgggcggg caagtccttc aaggtgggaa aaagatcatc gttgacgaac
     1561 gccaacaatt tatcccctgt aattccttca ttatcggcgg cccaattgcg ccataaatag
     1621 cgttctggca taggcagacg atacttttct tgttcaatct ccagtgcctg ttcctgtgca
     1681 tcaaagattt ttaaaaacag taaccaagaa agctggccca aacgctgggc atcaccatca
     1741 acaccggcat ctttgcgcat gatgtcttgc aagcttttaa tagcagatga gattgacata
     1801 tggctgccgc gcggcaccag gccgctgctg tgatgatgat gatgatggct gctgcccatg
     1861 gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca
     1921 caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc
     1981 ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc
     2041 gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc
     2101 gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca
     2161 ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg
     2221 caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt
     2281 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     2341 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     2401 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     2461 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     2521 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     2581 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     2641 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     2701 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     2761 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     2821 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     2881 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     2941 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     3001 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     3061 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     3121 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     3181 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     3241 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     3301 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     3361 cgattcatta atgcagctgg cacgacagtt tcccgactgg aaagcgggca gtgagcgcaa
     3421 cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag
     3481 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     3541 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat
     3601 tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt
     3661 cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg
     3721 cgagaagcag gccattatcg ccggcatggc ggccgacgcg ctgggctacg tcttgctggc
     3781 gttcgcgacg cgaggctgga tggccttccc cattatgatt cttctcgctt ccggcggcat
     3841 cgggatgccc gcgttgcagg ccatgctgtc caggcaggta gatgacgacc atcagggaca
     3901 gcttcaagga tcgctcgcgg ctcttaccag cctaacttcg atcattggac cgctgatcgt
     3961 cacggcgatt tatgccgcct cggcgagcac atggaacggg ttggcatgga ttgtaggcgc
     4021 cgccctatac cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc
     4081 gacctgaatg gaagccggcg gcacctcgct aacggattca ccactccaag aattggagcc
     4141 aatcaattct tgcggagaac tgtgaatgcg caaaccaacc cttggcagaa catatccatc
     4201 gcgtccgcca tctccagcag ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca
     4261 cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt
     4321 actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa
     4381 acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg
     4441 gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc
     4501 tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg
     4561 atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt
     4621 aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt
     4681 atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa
     4741 aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa
     4801 ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct
     4861 gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     4921 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     4981 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     5041 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag
     5101 tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     5161 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5221 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5281 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5341 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5401 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5461 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5521 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5581 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5641 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5701 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5761 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     5821 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5881 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5941 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6001 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6061 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6121 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6181 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6241 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6301 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6361 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc
     6421 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6481 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6541 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6601 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6661 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac
     6721 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6781 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6841 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6901 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6961 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7021 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     7081 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     7141 gcgtatcacg aggccctttc gtcttcaaga a
//
