LOCUS       sequence-439146-        6299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BsaI-associated switch methylase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6299)
  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 6299)
  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: POC1472
FEATURES             Location/Qualifiers
     source          1..6299
                     /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             1236..1258
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1273..1297
                     /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(1297..1316)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1298..1316)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1404..1423)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1629..1706
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1707..2789
                     /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(1726..1745)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3598..3789
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3783..3805)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3891..4030
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3946..3965
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4045..4062)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4216..4804)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4296..4315)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4915..4934)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4926..5741
                     /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             4926..5741
                     /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(4999..5018)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5833..6288)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5970..5991)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6182..6201
                     /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 gagttcccct tctttaaact
      181 cgtaaacacc gttcgattca aaataggcat ttgcctcatt caggcgggcc gctgctaact
      241 tgtaaaatgt gtcgtcaatt tctgcagcgt aactgcggcg accaagtaat gacgatgcca
      301 cactagccga gcacagacct ccaaatggtt cccagactgt atcgccgaca ttactggctg
      361 ctgctacctg agtaagcatc aggtccaggg gcttttggtt cagatgtgct gctgactgtt
      421 tggtgggttt atacgtgcga ggtgcggagc gttccattgt ccctttcaaa cgctcactat
      481 cggccaacgg cggacggctc cacacgtttg tgactccatt gcggtgattc catgtagtgc
      541 gtaaactatc ccaatcatga gctgaaatca tagttttgcc atccagactg aaatagggcc
      601 aagaagtttt tttaccatat tgcatgcagt actccgccat cttttggacc gcgtcaccag
      661 gcggccagta ccacagccag tctgctgtca aatatttacg ggtcgcagcg ttcttcacac
      721 cacacgcttc attcgacttt gaaagggaca ggcccgagcg acgccactct gcgcgcaacc
      781 atgactttgc gtttaatgta agtccgtctc cagtttccag gtacaccgta cggcgataaa
      841 gggccgccac ctctgttacc acgggcaatt gacggattgt tttcccgtta acattgccgg
      901 caatgtgcgc cagtccttta tcccagacag ctaactgcac gtattcccat ccggtggaca
      961 ataataaagg gtggaccgtg gcccagccta cttctgtgtt ccaaaaccac aaagaagtgg
     1021 aaggcttggc cgctttggcc cacgccagca cgtgtggctt ataccagtcc gtcaacccgg
     1081 cagcgtcaac ggtgtccccg cgaaatccac gaacaccgta tgccccatcc gatacaatta
     1141 agtccggact aggccagcgc ccgtagactt ccgatacgtt acccttataa aggtgggaaa
     1201 gcttaaggtt tgtgtccttc tttgccatat gtatatctcc ttcttaaagt taaacaaaat
     1261 tatttctaga ggggaattgt tatccgctca caattcccct atagtgagtc gtattaattt
     1321 cgcgggatcg agatcgatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg
     1381 cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc
     1441 tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc
     1501 cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa
     1561 cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg
     1621 agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     1681 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
     1741 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     1801 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     1861 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     1921 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     1981 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
     2041 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
     2101 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
     2161 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
     2221 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
     2281 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
     2341 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
     2401 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
     2461 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggacatc tcggtagtgg
     2521 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     2581 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     2641 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     2701 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2761 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact
     2821 cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc
     2881 cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa
     2941 ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg
     3001 agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa
     3061 gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc
     3121 atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg
     3181 cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac
     3241 tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt
     3301 tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc
     3361 gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat
     3421 tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca
     3481 caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct
     3541 cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg
     3601 accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg
     3661 cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt
     3721 cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa
     3781 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     3841 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     3901 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     3961 ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     4021 ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4081 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4141 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4201 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4261 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4321 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4381 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4441 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4501 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4561 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4621 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     4681 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4741 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4801 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4861 ttaagggatt ttggtcatga acaataaaac tgtctgctta cataaacagt aatacaaggg
     4921 gtgttatgag ccatattcaa cgggaaacgt cttgctctag gccgcgatta aattccaaca
     4981 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     5041 caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     5101 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     5161 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     5221 ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     5281 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     5341 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     5401 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     5461 ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt
     5521 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     5581 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     5641 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     5701 ataaattgca gtttcatttg atgctcgatg agtttttcta agaattaatt catgagcgga
     5761 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5821 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     5881 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     5941 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     6001 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     6061 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     6121 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     6181 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     6241 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
