LOCUS       sequence-439182-        6437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BsaI-associated non-switchable methylase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6437)
  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 6437)
  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: POC1464
FEATURES             Location/Qualifiers
     source          1..6437
                     /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             1374..1396
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1411..1435
                     /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(1435..1454)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1436..1454)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1542..1561)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1767..1844
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1845..2927
                     /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(1864..1883)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3736..3927
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3921..3943)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4029..4168
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4084..4103
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4183..4200)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4354..4942)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4434..4453)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5053..5072)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5064..5879
                     /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             5064..5879
                     /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(5137..5156)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5971..6426)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6108..6129)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6320..6339
                     /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 gcaagcttaa
      181 gcgacggctt aatgttgcgt tcataatact ctgagggtgt aatgtttcca cgcataaaca
      241 actggtaatc attttccggg tggtggatat agtatttacc agatccatcc tcgccgtgtt
      301 gtaatggggg aaggtcagat aacgcatctt ttactgacac ccaagactct ttatcttccg
      361 tcagcttctg aggtggttcg atcgagaaca gcggatcatt gctgcccaca agaattacac
      421 gcttacggcg ttgtggaatt gcgtaatgtt ccgaatttaa aatccaacca ttcatggttt
      481 tcattgtgga cgagaattcc tccttaacac gttcaaagac ctttcctttt tccatgttca
      541 acaggccggc gacgttctcg aaaatgaatc cattcggctt aatctcgtta agcagcgact
      601 tgtagtgcat aaacagactg ttgcgtgggt cgtccattgt acgcgcgttg ccagccgtgc
      661 taaatccctg gcacggaggc ccgcccagaa tccacagctg tttatcaaaa tataatttct
      721 taaagccaga aatcttgtct gcgatcttag aaaaagtttc gttgtccgaa atgcttccgc
      781 acaaaacttc tttgtgtacg ttgttcgagt atgtctgtaa gtacttttcc tcaatgtcat
      841 tagctaacaa tggagtccac ccggcccact taaagcccaa ccccaatccg cctgcaccac
      901 tgaataagtc aatgtagtag ccggtattac cgatcgcttt ttcaatttct tttgcaataa
      961 gaaacgcaag aaagggaggc acggcattac cgatttgcgt attaatcgcc gtttgacctc
     1021 caaaaaagat aaagtcatca gggaacgact gcagacgtgc agcttcgcgt tgtgaaagta
     1081 cgcgatcttg ctcgtagtga atgtggcatc cgttgccagg acgattgaaa taagtattga
     1141 tcgtataagc tggcatatcg ggcaataaac gcccgtagta tgtgctgcgc gacccttttc
     1201 cttgagcata gctgtcacga atctgttcga tacgtttcga tggtacatcc aagggaatat
     1261 tcttccagtt tcccccaggc gggacagagt taacgatcat catatccagc tcacttaatt
     1321 tggcggcctt gtggtttgga atcatgaatg tttcagactt ggtcatatgt atatctcctt
     1381 cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat
     1441 agtgagtcgt attaatttcg cgggatcgag atcgatctcg atcctctacg ccggacgcat
     1501 cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac
     1561 cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat
     1621 ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct
     1681 tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc
     1741 gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta
     1801 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     1861 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     1921 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     1981 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     2041 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     2101 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     2161 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     2221 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     2281 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     2341 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     2401 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     2461 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     2521 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     2581 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     2641 cggacatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     2701 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     2761 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     2821 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     2881 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     2941 aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc
     3001 aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact
     3061 gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc
     3121 gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc
     3181 ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag
     3241 caggccatta tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg
     3301 aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc
     3361 gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg
     3421 gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt
     3481 atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta
     3541 ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc
     3601 gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt
     3661 atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct
     3721 tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc
     3781 agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca
     3841 gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt
     3901 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     3961 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     4021 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact
     4081 atgcggcatc agagcagatt gtactgagag tgcaccattg cggtgtgaaa taccgcacag
     4141 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct
     4201 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4261 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4321 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4381 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4441 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4501 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4561 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4621 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4681 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4741 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     4801 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4861 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4921 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4981 gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca
     5041 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc
     5101 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     5161 tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt
     5221 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
     5281 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     5341 atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat
     5401 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     5461 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
     5521 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     5581 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
     5641 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     5701 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     5761 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     5821 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag
     5881 aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5941 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
     6001 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     6061 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     6121 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     6181 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     6241 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     6301 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     6361 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     6421 gcgcgtccca ttcgcca
//
