LOCUS       sequence-439149-        6701 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 6701)
  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 6701)
  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: POC1476
FEATURES             Location/Qualifiers
     source          1..6701
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1638..1660
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1675..1699
                     /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(1699..1718)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1700..1718)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1806..1825)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2031..2108
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2109..3191
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2128..2147)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4000..4191
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4185..4207)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4293..4432
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4348..4367
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4447..4464)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4618..5206)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4698..4717)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5317..5336)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5328..6143
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             5328..6143
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5401..5420)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6235..6690)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6372..6393)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6584..6603
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gaggttcgcc tctttaccac
      181 tcaacgcagt tcctaagatg tcacgaagct gatggcgaag agtttggatg gcctcttgct
      241 gtttggcgta ttgtgctaac agctcgtctg ggtcatggga tacggtttct gcctggtggg
      301 ggttcttaat atcaagattg aagttacggg cgataacttc atcaatagag actttccagg
      361 cctgctcatt ctcgacgcga cttgcaaacc catccgcttc gttaccccac caatcaattt
      421 ctgcctgaaa ctcctcaaat ttcattggtt tcgtcttgct gtagttttta acacctgcgg
      481 ggtaagggtg ttcataaaac cagatctcct tggtcggctg ccctttcgtg aaaaataaca
      541 ggttagtttt gatccccgta taggggttaa aaacgccgtt aggtaaacgg acaattgtgt
      601 gcaaattgca ctcttcggtc agcaactttt taatcttagt tttaacacct tcaccaaaaa
      661 gcgtaccatc gggcaacacc acggcggcac gaccgttctt agctaagacc tcaataataa
      721 gctgaagaaa taagtccgcc gtctcacgtg tctgcatgtc agaggggaag ttcttttcaa
      781 tgccatcttc ctcagtaccc ccgaatgggg ggttagtgat gataacatcc atttgctcgt
      841 cccacgatga aagtggttta ttaagagtat tgtcgtggcg gatttgaacg gggacttcga
      901 tgccgtgaag cagcatgttg gtcgtacaca acaagtgtgg taattgcttc ttttcgaccc
      961 catgaatttg tttctgcaag atttgatggt cagtcacagt atgggcataa tggtttttaa
     1021 cgtggtcgaa ggcgcatgcc aaaaatcccc ccgtaccaca agccggatcc atgatcgact
     1081 cacccagttt agggtctaca cgttcaacca tgaagcgagt aacagcgcga ggcgtataaa
     1141 attcaccagc attaccggca gcctgcaagt cacgcaaaat ttgctcgtag atatctccaa
     1201 aaagatggcg ctctgaagca cgtgtaaagt caatctcatt taacttgttg ataacttggc
     1261 gcagcaacgt cccgtttttc atgtagttat aagcgtcgga gaaagcctgc tttactacat
     1321 acccgcgggg gttgatgtcg atctgggcgg gcaagtcctt caaggtggga aaaagatcat
     1381 cgttgacgaa cgccaacaat ttatcccctg taattccttc attatcggcg gcccaattgc
     1441 gccataaata gcgttctggc ataggcagac gatacttttc ttgttcaatc tccagtgcct
     1501 gttcctgtgc atcaaagatt tttaaaaaca gtaaccaaga aagctggccc aaacgctggg
     1561 catcaccatc aacaccggca tctttgcgca tgatgtcttg caagctttta atagcagatg
     1621 agattgacat atgtatatct ccttcttaaa gttaaacaaa attatttcta gaggggaatt
     1681 gttatccgct cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgagatcgat
     1741 ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc
     1801 tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat
     1861 gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc
     1921 catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact
     1981 gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg
     2041 aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
     2101 tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc
     2161 agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag
     2221 tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg
     2281 gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc
     2341 aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga
     2401 tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac
     2461 gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag
     2521 ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca
     2581 gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg
     2641 gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc
     2701 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
     2761 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
     2821 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
     2881 ccgagtccgg gctgcgcgtt ggtgcggaca tctcggtagt gggatacgac gataccgaag
     2941 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
     3001 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
     3061 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct
     3121 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     3181 gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct
     3241 cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg
     3301 actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg
     3361 gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc
     3421 ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc
     3481 gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc cccacgggtg
     3541 cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt
     3601 tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct
     3661 gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg
     3721 cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta
     3781 ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt
     3841 ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg
     3901 gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt
     3961 acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc
     4021 gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac
     4081 gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag
     4141 ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     4201 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     4261 ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat
     4321 agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     4381 attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
     4441 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4501 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4561 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4621 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4681 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4741 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4801 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4861 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4921 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4981 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5041 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5101 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5161 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5221 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5281 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     5341 aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg
     5401 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg
     5461 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     5521 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     5581 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa
     5641 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     5701 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     5761 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     5821 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac
     5881 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     5941 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     6001 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     6061 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     6121 tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt
     6181 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg
     6241 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     6301 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     6361 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     6421 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     6481 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     6541 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     6601 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     6661 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc a
//
