LOCUS       sequence-437783-        7475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BsaI-associated switch methylase subunit S.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7475)
  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 7475)
  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: POC1477
FEATURES             Location/Qualifiers
     source          1..7475
                     /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(2107..2124)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2134..2151)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2170..2192
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2207..2231
                     /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(2231..2250)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2232..2250)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2334..2353)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2559..2636
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(2656..2675)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5030..5221
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5215..5237)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5323..5462
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5378..5397
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5477..5494)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5648..6236)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5728..5747)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6407..7198,7199..7267))
                     /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     7030..7049
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7268..7372)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7440..7458
                     /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 atccttagtt aattgcggca tcagtcaggg cgtccgcaag
      361 atgaagctgc gtctgttgag cgctctgaat ctgcagcttt aatttttcac acagttcatt
      421 aaaatcgttt aatttaagca tgattttttc tgcttcgcta ctaggtggaa tagccactgg
      481 acatccacga agttgcgtct tgttgatact cgccaggttg gtagtttgtt tagaggcact
      541 cgcgaagtaa tcacgggcgt aaggagagtt catgtacgtc tcaagccaat agggatcgat
      601 gtcctgcccg ataatacgtc ccttaaacac gtggttttga ttggcgatat accagtcgtg
      661 gcaccaaaca gtagtacgcc ctactgtgtc ccaatcgccg ccttccgtga ttaacaagtc
      721 gcccttgatc acgtggtact tatccttctc ttcttcggga atgtcgattt ctttaacttc
      781 tgaaaggtcc aaataaccgc gctgcacgtt ggctacgcgc aaatacggga tagaaataag
      841 cttgcgattt gctaagttgc ggcccttggt aatgcccgat tgaatatcta cgacgtcctc
      901 aaataaacac cattcccaac ccgatggtaa ctcgaatggt ttttcctcat cactaatcgg
      961 cggtaatggc ttttgcttct tcatctttcc atcctttacc aactgtgctt tttcctgtgc
     1021 aatacgcttc agcagttcag atgctggttc atcgtttggg tcctgtggca ccagcttacc
     1081 catgacagcc agttgcagga tcgtctgctt cagggcgtcg atactgactt cagttgtaaa
     1141 aagagtatcg aagtgttcgc tgatacgggc ccaattttca gctaactcat ctgcattctg
     1201 gctgtccgtc aaggtggtta acaacgtttc cactaattgt tgatgtgcat caagggaagt
     1261 caggctatat tgttctaact gatcgcaaag ggacatcagc tccttaattt ttttgaagat
     1321 actatattgt tcctggacgg gtggaattgg caaaagcaat gttttcaggt gcccggttgc
     1381 gatgcggaag cgcgtcgcgc cctttccaaa cgattctact tgctgacgaa atgttaaact
     1441 gttaatggcg gaaataatat attccgcact ataatttcct gaagggcgca ttgtagcaat
     1501 atctactaca gtgatagcgc tttgggaaag cttaggaaag atgcacgcgc gtccgatcgg
     1561 gttcggtaag cgcgcgataa ggatatcacc ctccatcagt tgatggcagt tcaaacgatg
     1621 aaacgtcgaa gtgttgatat agcgatcgga cttgtccttg aaatctccca cgccaacatc
     1681 agctaactga atcaaacggt aagcgcctga cgggtcctgg tcttttgact cgatccaatc
     1741 cccgtcaatg attcccatag gagctaattc atttaaacga gcccattccc agcccgctgg
     1801 cagttcaaat ggcttttcct cctcagagat ttctggcaag ggcttctgct ttttaatctt
     1861 accttgtttc accagctccg ctttttctgt cgcgatgcgt tctaaaagca ccgaagcggg
     1921 cttgtcattt gggtcttgcg ggaccagctt gccacgtacg gccagttcta aaataagttc
     1981 gcgaagcttc ttaattccat acaagtcgat tttgccgctg ctcccgcgac cggccgtgga
     2041 gcgcgtctga agggcggtag tccaggtgtc gatatgatct acaataagtt tttccaccgc
     2101 catatggctg ccgcgcggca ccaggccgct gctgtgatga tgatgatgat ggctgctgcc
     2161 catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg
     2221 ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta
     2281 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat
     2341 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt
     2401 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca
     2461 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct
     2521 aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa
     2581 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     2641 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     2701 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     2761 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     2821 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     2881 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     2941 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     3001 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     3061 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     3121 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     3181 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     3241 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     3301 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     3361 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     3421 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     3481 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     3541 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     3601 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     3661 tggccgattc attaatgcag ctggcacgac agtttcccga ctggaaagcg ggcagtgagc
     3721 gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct
     3781 tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg
     3841 cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg
     3901 tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg
     3961 tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt
     4021 tcggcgagaa gcaggccatt atcgccggca tggcggccga cgcgctgggc tacgtcttgc
     4081 tggcgttcgc gacgcgaggc tggatggcct tccccattat gattcttctc gcttccggcg
     4141 gcatcgggat gcccgcgttg caggccatgc tgtccaggca ggtagatgac gaccatcagg
     4201 gacagcttca aggatcgctc gcggctctta ccagcctaac ttcgatcatt ggaccgctga
     4261 tcgtcacggc gatttatgcc gcctcggcga gcacatggaa cgggttggca tggattgtag
     4321 gcgccgccct ataccttgtc tgcctccccg cgttgcgtcg cggtgcatgg agccgggcca
     4381 cctcgacctg aatggaagcc ggcggcacct cgctaacgga ttcaccactc caagaattgg
     4441 agccaatcaa ttcttgcgga gaactgtgaa tgcgcaaacc aacccttggc agaacatatc
     4501 catcgcgtcc gccatctcca gcagccgcac gcggcgcatc tcgggcagcg ttgggtcctg
     4561 gccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg
     4621 ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg
     4681 caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag
     4741 tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg
     4801 ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg
     4861 agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc
     4921 cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat
     4981 cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca
     5041 ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga
     5101 gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca
     5161 cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     5221 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     5281 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     5341 acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
     5401 agagtgcacc attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     5461 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5521 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5581 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5641 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5701 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5761 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5821 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5881 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5941 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6001 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6061 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     6121 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6181 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6241 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6301 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6361 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6421 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6481 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6541 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6601 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6661 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6721 ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6781 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6841 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6901 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6961 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7021 caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7081 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7141 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7201 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7261 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7321 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7381 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     7441 ataggcgtat cacgaggccc tttcgtcttc aagaa
//
