LOCUS       sequence-439180-        7010 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 7010)
  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 7010)
  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: POC1478
FEATURES             Location/Qualifiers
     source          1..7010
                     /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             1947..1969
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1984..2008
                     /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(2008..2027)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2009..2027)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2115..2134)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2340..2417
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2418..3500
                     /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(2437..2456)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4309..4500
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4494..4516)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4602..4741
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4657..4676
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4756..4773)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4927..5515)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5007..5026)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5626..5645)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5637..6452
                     /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             5637..6452
                     /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(5710..5729)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6544..6999)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6681..6702)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6893..6912
                     /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 gaggttaatt gcggcatcag
      181 tcagggcgtc cgcaagatga agctgcgtct gttgagcgct ctgaatctgc agctttaatt
      241 tttcacacag ttcattaaaa tcgtttaatt taagcatgat tttttctgct tcgctactag
      301 gtggaatagc cactggacat ccacgaagtt gcgtcttgtt gatactcgcc aggttggtag
      361 tttgtttaga ggcactcgcg aagtaatcac gggcgtaagg agagttcatg tacgtctcaa
      421 gccaataggg atcgatgtcc tgcccgataa tacgtccctt aaacacgtgg ttttgattgg
      481 cgatatacca gtcgtggcac caaacagtag tacgccctac tgtgtcccaa tcgccgcctt
      541 ccgtgattaa caagtcgccc ttgatcacgt ggtacttatc cttctcttct tcgggaatgt
      601 cgatttcttt aacttctgaa aggtccaaat aaccgcgctg cacgttggct acgcgcaaat
      661 acgggataga aataagcttg cgatttgcta agttgcggcc cttggtaatg cccgattgaa
      721 tatctacgac gtcctcaaat aaacaccatt cccaacccga tggtaactcg aatggttttt
      781 cctcatcact aatcggcggt aatggctttt gcttcttcat ctttccatcc tttaccaact
      841 gtgctttttc ctgtgcaata cgcttcagca gttcagatgc tggttcatcg tttgggtcct
      901 gtggcaccag cttacccatg acagccagtt gcaggatcgt ctgcttcagg gcgtcgatac
      961 tgacttcagt tgtaaaaaga gtatcgaagt gttcgctgat acgggcccaa ttttcagcta
     1021 actcatctgc attctggctg tccgtcaagg tggttaacaa cgtttccact aattgttgat
     1081 gtgcatcaag ggaagtcagg ctatattgtt ctaactgatc gcaaagggac atcagctcct
     1141 taattttttt gaagatacta tattgttcct ggacgggtgg aattggcaaa agcaatgttt
     1201 tcaggtgccc ggttgcgatg cggaagcgcg tcgcgccctt tccaaacgat tctacttgct
     1261 gacgaaatgt taaactgtta atggcggaaa taatatattc cgcactataa tttcctgaag
     1321 ggcgcattgt agcaatatct actacagtga tagcgctttg ggaaagctta ggaaagatgc
     1381 acgcgcgtcc gatcgggttc ggtaagcgcg cgataaggat atcaccctcc atcagttgat
     1441 ggcagttcaa acgatgaaac gtcgaagtgt tgatatagcg atcggacttg tccttgaaat
     1501 ctcccacgcc aacatcagct aactgaatca aacggtaagc gcctgacggg tcctggtctt
     1561 ttgactcgat ccaatccccg tcaatgattc ccataggagc taattcattt aaacgagccc
     1621 attcccagcc cgctggcagt tcaaatggct tttcctcctc agagatttct ggcaagggct
     1681 tctgcttttt aatcttacct tgtttcacca gctccgcttt ttctgtcgcg atgcgttcta
     1741 aaagcaccga agcgggcttg tcatttgggt cttgcgggac cagcttgcca cgtacggcca
     1801 gttctaaaat aagttcgcga agcttcttaa ttccatacaa gtcgattttg ccgctgctcc
     1861 cgcgaccggc cgtggagcgc gtctgaaggg cggtagtcca ggtgtcgata tgatctacaa
     1921 taagtttttc caccgccata tgtatatctc cttcttaaag ttaaacaaaa ttatttctag
     1981 aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc
     2041 gagatcgatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg
     2101 tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt
     2161 cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact
     2221 gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa
     2281 cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gagatcccgg
     2341 acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
     2401 aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
     2461 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
     2521 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
     2581 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
     2641 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
     2701 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
     2761 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
     2821 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
     2881 ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg
     2941 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
     3001 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
     3061 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
     3121 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
     3181 gcgccattac cgagtccggg ctgcgcgttg gtgcggacat ctcggtagtg ggatacgacg
     3241 ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc
     3301 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
     3361 gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
     3421 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     3481 gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca
     3541 ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg
     3601 cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga
     3661 caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg
     3721 atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc
     3781 actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc
     3841 ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc
     3901 cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc
     3961 aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt
     4021 ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc
     4081 tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga
     4141 gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc
     4201 agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc
     4261 ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag
     4321 gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag
     4381 aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac
     4441 gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     4501 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     4561 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     4621 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga
     4681 gagtgcacca ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     4741 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4801 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4861 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4921 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4981 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5041 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5101 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5161 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5221 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5281 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5341 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     5401 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5461 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5521 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5581 tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
     5641 gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg
     5701 atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc
     5761 gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg
     5821 ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc
     5881 cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc
     5941 ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg
     6001 atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta
     6061 acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg
     6121 atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa
     6181 tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg
     6241 ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa
     6301 tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt
     6361 cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc
     6421 agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg atacatattt
     6481 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6541 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     6601 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     6661 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     6721 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     6781 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     6841 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     6901 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     6961 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
