LOCUS       sequence-437746-        6347 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 6347)
  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 6347)
  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: POC1473
FEATURES             Location/Qualifiers
     source          1..6347
                     /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(979..996)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1006..1023)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1042..1064
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1079..1103
                     /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(1103..1122)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1104..1122)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1206..1225)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1431..1508
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(1528..1547)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3902..4093
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4087..4109)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4195..4334
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4250..4269
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4349..4366)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4520..5108)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4600..4619)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5279..6070,6071..6139))
                     /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     5902..5921
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6140..6244)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6312..6330
                     /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 atccttattc gatgatacag tagccagggt ggccaaagga
      361 aatgcgtgct tcgggcaagt tgttcttaat cttctcgatg gactgctggt aataatcaga
      421 gttaatctca cacgctgtta aatcaaattg ggcaatacaa catgcaatcg ctaaagtccc
      481 agatcccagg tgtgtatcca aaattttatc accctgtttt gcgtacatct tcaataacca
      541 ttggtaaagc tctactggct tctgcgtcgg atgtgtcttg ttgcggtttt tacgaacact
      601 gaagcgaaaa atcttcgatg ggcgatcgaa acttgaccag gccatttctg ccatcgaaaa
      661 attgtttaag gtttccggtt ggttcttgtc ccagataatg aagcccttat tatattcgct
      721 acgtaaccac aattctccaa aatagttgcc cccccaaata atctgattct tgctaacgcg
      781 aaataactcg ttgaagtagg tctggtcagg tttaatatcc cactggctgt attcgttcat
      841 gttgaattta taatctccac cgcgtttagt tttgttcaag attccataag gggggtcaac
      901 aatggccagg tcaaagtact tgtctggata gcgactcatc aggatcatat tatcttcgtt
      961 tgagattgtg atcatatggc tgccgcgcgg caccaggccg ctgctgtgat gatgatgatg
     1021 atggctgctg cccatggtat atctccttct taaagttaaa caaaattatt tctagagggg
     1081 aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat
     1141 ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc
     1201 tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat
     1261 gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc
     1321 catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact
     1381 gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg
     1441 aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
     1501 tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc
     1561 agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag
     1621 tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg
     1681 gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc
     1741 aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga
     1801 tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac
     1861 gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag
     1921 ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca
     1981 gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg
     2041 gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc
     2101 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
     2161 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
     2221 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
     2281 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag
     2341 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
     2401 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
     2461 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct
     2521 ctccccgcgc gttggccgat tcattaatgc agctggcacg acagtttccc gactggaaag
     2581 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc
     2641 gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga
     2701 ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg
     2761 cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc
     2821 tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg
     2881 ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc gacgcgctgg
     2941 gctacgtctt gctggcgttc gcgacgcgag gctggatggc cttccccatt atgattcttc
     3001 tcgcttccgg cggcatcggg atgcccgcgt tgcaggccat gctgtccagg caggtagatg
     3061 acgaccatca gggacagctt caaggatcgc tcgcggctct taccagccta acttcgatca
     3121 ttggaccgct gatcgtcacg gcgatttatg ccgcctcggc gagcacatgg aacgggttgg
     3181 catggattgt aggcgccgcc ctataccttg tctgcctccc cgcgttgcgt cgcggtgcat
     3241 ggagccgggc cacctcgacc tgaatggaag ccggcggcac ctcgctaacg gattcaccac
     3301 tccaagaatt ggagccaatc aattcttgcg gagaactgtg aatgcgcaaa ccaacccttg
     3361 gcagaacata tccatcgcgt ccgccatctc cagcagccgc acgcggcgca tctcgggcag
     3421 cgttgggtcc tggccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg
     3481 ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag
     3541 cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg
     3601 tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg
     3661 catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg
     3721 gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc
     3781 ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct
     3841 ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc
     3901 agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt
     3961 aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc
     4021 gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg
     4081 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     4141 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     4201 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     4261 cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa
     4321 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4381 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4441 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4501 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4561 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4621 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4681 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4741 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4801 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4861 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4921 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     4981 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5041 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5101 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5161 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5221 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5281 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5341 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5401 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5461 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5521 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5581 ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5641 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5701 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5761 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5821 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5881 cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5941 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6001 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6061 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6121 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6181 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6241 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     6301 taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaa
//
