LOCUS       sequence-439179-        5897 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BpiI-associated switch methylase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5897)
  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 5897)
  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: POC1474
FEATURES             Location/Qualifiers
     source          1..5897
                     /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             834..856
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    871..895
                     /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(895..914)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(896..914)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1002..1021)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1227..1304
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1305..2387
                     /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(1324..1343)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3196..3387
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3381..3403)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3489..3628
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3544..3563
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3643..3660)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3814..4402)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3894..3913)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4513..4532)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4524..5339
                     /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             4524..5339
                     /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(4597..4616)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5431..5886)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5568..5589)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5780..5799
                     /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 gagtgcggcc gcaagctttt
      181 cgatgataca gtagccaggg tggccaaagg aaatgcgtgc ttcgggcaag ttgttcttaa
      241 tcttctcgat ggactgctgg taataatcag agttaatctc acacgctgtt aaatcaaatt
      301 gggcaataca acatgcaatc gctaaagtcc cagatcccag gtgtgtatcc aaaattttat
      361 caccctgttt tgcgtacatc ttcaataacc attggtaaag ctctactggc ttctgcgtcg
      421 gatgtgtctt gttgcggttt ttacgaacac tgaagcgaaa aatcttcgat gggcgatcga
      481 aacttgacca ggccatttct gccatcgaaa aattgtttaa ggtttccggt tggttcttgt
      541 cccagataat gaagccctta ttatattcgc tacgtaacca caattctcca aaatagttgc
      601 ccccccaaat aatctgattc ttgctaacgc gaaataactc gttgaagtag gtctggtcag
      661 gtttaatatc ccactggctg tattcgttca tgttgaattt ataatctcca ccgcgtttag
      721 ttttgttcaa gattccataa ggggggtcaa caatggccag gtcaaagtac ttgtctggat
      781 agcgactcat caggatcata ttatcttcgt ttgagattgt gatcatatgt atatctcctt
      841 cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat
      901 agtgagtcgt attaatttcg cgggatcgag atcgatctcg atcctctacg ccggacgcat
      961 cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac
     1021 cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat
     1081 ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct
     1141 tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc
     1201 gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta
     1261 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     1321 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     1381 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     1441 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     1501 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     1561 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     1621 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     1681 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     1741 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     1801 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     1861 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     1921 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     1981 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     2041 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     2101 cggacatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     2161 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     2221 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     2281 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     2341 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     2401 aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc
     2461 aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact
     2521 gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc
     2581 gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc
     2641 ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag
     2701 caggccatta tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg
     2761 aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc
     2821 gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg
     2881 gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt
     2941 atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta
     3001 ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc
     3061 gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt
     3121 atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct
     3181 tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc
     3241 agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca
     3301 gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt
     3361 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     3421 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     3481 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact
     3541 atgcggcatc agagcagatt gtactgagag tgcaccattg cggtgtgaaa taccgcacag
     3601 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct
     3661 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3721 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3781 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3841 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3901 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3961 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4021 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4081 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4141 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4201 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     4261 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4321 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4381 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4441 gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca
     4501 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc
     4561 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     4621 tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt
     4681 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
     4741 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     4801 atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat
     4861 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     4921 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
     4981 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     5041 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
     5101 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     5161 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     5221 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     5281 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag
     5341 aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5401 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
     5461 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     5521 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     5581 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     5641 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     5701 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     5761 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     5821 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     5881 gcgcgtccca ttcgcca
//
