LOCUS       sequence-439158-        6882 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 6882)
  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 6882)
  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: POC1480
FEATURES             Location/Qualifiers
     source          1..6882
                     /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             1823..1845
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1860..1884
                     /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(1884..1903)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1885..1903)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1987..2006)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2212..2289
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2290..3372
                     /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(2309..2328)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4181..4372
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4366..4388)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4474..4613
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4529..4548
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4628..4645)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4799..5387)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4879..4898)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5498..5517)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5509..6324
                     /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             5509..6324
                     /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(5582..5601)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6416..6871)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6553..6574)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6765..6784
                     /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 gcaagcttat
      181 cgtagtgctg gaagatgcgt aacaaagact ctttttctgc caactcgatt ttgtcgaagc
      241 tactaagcaa tttgttgaac tcattcaggt caagtttgta aatgttcttc gccacatatg
      301 cttcaatctt taattccgca tcgcagcacc cctcaatgca gctaattacc agctgtttca
      361 actcactgtg ttgcagtaag atcttttcgc tagggatcgc ggttttacga agagcagaca
      421 gactaacatg cccagtagcc aaaaaggagc gcaattgaaa ttcgaagcac agagagttaa
      481 taattccaag caaggaaaac agggaatcct gggacccgga tttgtagtat actacaccca
      541 atgagttacc agccaacgag ttaggcggaa tgatagttgc gatcatgcgg cgtttttggc
      601 ttgggcgcga aatatcgcgc caggcgatgc ggacgaagtt gctattggaa agcaccttgt
      661 caatcttttt cgtaatgcgc agagactttt cattgtggaa attgtagcgg ccaatcattt
      721 tgcccttaat aaacaacagc cccccacccg acttctgagt ccaatccgac gagccagttt
      781 cgtcgatttc acggcccgcc caaatctcgc cgttcttttc taaactaccc cacgttgggt
      841 aaccctcttg aagtttgcgc aaaagatgga ccgcatgcac gccttgagac acggggatcg
      901 acagatattg ggaatcgatg cttgacaaat cgaaaaaacc caatgacttg atgttcaaat
      961 ttttatcgta gacgcttaat tgaatattat cattcaagga ttctcccttg ttaaaaatga
     1021 aggagcagga gctcacatct gctccaccga aaagttttgc ttcggctgga taatagttaa
     1081 tacagtcgat acgtccggaa ttgaaaaact tctcacgtaa aatataggat tgctcgtccg
     1141 caaaaaagct ggcgggcatg acgatcgcta ctaacccatt tttgttgcaa atctcaagag
     1201 ataactctgc accaacacga cttaagttcg taccccagcc agcgaacttg cgcttcggct
     1261 gactataggg aagcacacgt gaaaggtaat catcaaattc acgaaggctg tcaatataca
     1321 tggatttcat gcttggttcg aaaaaatcaa gctcacgaga atcaggttta atgttctccc
     1381 agggaggatt tgtgatcaca cagtcgaagt agtctacata cttcttaaat tcgtaaaata
     1441 catccgagtt cttaattgta atattgatat tagcgccggc taacttcaat tccgacagag
     1501 cgtttttggc gtaagtcaga ccggtgttct caatgtcaaa caggtagacg ttccattcga
     1561 catcggggaa gccgttaatc atccactgct tgatcaaaag agtgatcaga cgaccgtcac
     1621 ctgaaaaggg atcagcaacg ttatagctct ttttatcaca ggagttaatc agcggaagta
     1681 acttctcgat cattaacagc gcgattttgg ggtcggtgta gtatttaccg gtagcttttt
     1741 tcgtgaactt agtagtttcg tccagcgaca ggctgttgat taaatggatg tgtttcatat
     1801 agaagatctc ctccatggta tatctccttc ttaaagttaa acaaaattat ttctagaggg
     1861 gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga
     1921 tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg
     1981 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca
     2041 tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg
     2101 ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac
     2161 tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc ggacaccatc
     2221 gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     2281 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     2341 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     2401 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     2461 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     2521 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     2581 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     2641 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     2701 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     2761 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     2821 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     2881 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     2941 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     3001 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     3061 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
     3121 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     3181 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     3241 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     3301 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     3361 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc
     3421 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     3481 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
     3541 ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg
     3601 cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc
     3661 cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt
     3721 gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg
     3781 ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc
     3841 tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac
     3901 gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct
     3961 accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt
     4021 tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg
     4081 ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat
     4141 tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac
     4201 cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa
     4261 cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga
     4321 gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     4381 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     4441 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga
     4501 tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
     4561 cattgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt
     4621 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4681 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4741 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4801 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4861 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4921 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4981 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5041 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5101 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5161 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5221 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5281 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5341 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5401 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5461 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     5521 caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat
     5581 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat
     5641 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     5701 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     5761 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa
     5821 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     5881 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     5941 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     6001 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     6061 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     6121 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     6181 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     6241 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     6301 ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat
     6361 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
     6421 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     6481 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     6541 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     6601 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     6661 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     6721 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     6781 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     6841 gccgcgctta atgcgccgct acagggcgcg tcccattcgc ca
//
