LOCUS       sequence-439145-        6392 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 6392)
  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 6392)
  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: POC1470
FEATURES             Location/Qualifiers
     source          1..6392
                     /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             1329..1351
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1366..1390
                     /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(1390..1409)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1391..1409)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1497..1516)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1722..1799
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1800..2882
                     /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(1819..1838)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3691..3882
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3876..3898)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3984..4123
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4039..4058
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4138..4155)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4309..4897)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4389..4408)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5008..5027)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5019..5834
                     /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             5019..5834
                     /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(5092..5111)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5926..6381)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6063..6084)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6275..6294
                     /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 tgatagctaa ggaggacagc atctcgatat actgctgggc tgtgatcttc ttgcgcatga
      241 atgactgata aatattttgt ggctcacaaa cgtagttctt ccaagagccg tcctcattag
      301 gcgacaaaag gggtaagtct gaaagggcct cctttacgga aatggcactg cttaaggcgt
      361 caaacagggt cttttctcct ttaagagacg taattgggat agggggctcg ctgttcttcg
      421 ttccggtgga gtccccgatg atgactacgc ggttgcggcg ttggggcaca ccgtaatcca
      481 ccgaattaag tttatatacg aaaagattcg acaccgtctt agacaattcc gatttaacca
      541 tctcaaagaa tgctcccttc tccatcgaca acaggccagt cacgttttca aaaataaaac
      601 cgtctggctt gacttctttc aggatactca cgtaactttc gaacagccag ttgcgctcgt
      661 ccacgattga acgttttttt cctgcagtcg aaaacccttg gcagggtggt cccccaagaa
      721 caaacaatgg acggtccggg ttcgacttct ttgcgatcag gcacttttga atgatagtct
      781 cactgacctt ttcgtcgcgg atatccccaa gcactacttc attgtgaata ttattcatgt
      841 aggtggtcag cgcccattta tcgatgtcat ttgcgatgat cggtttccac ccggcccaaa
      901 ggaaacctaa ggaaaggccc cccgccccac taaaaagatc gacaaattgg cctttgaagg
      961 gaaatgcctt ggcgatctga taagcaagaa ggggcggaac agcgttgccg atctggttgt
     1021 tgatggccgt cttgctaccg tagaagatga aatcgtcagg gaaagactgc aagcgcgcgg
     1081 cctcacgctg tgacagagtg cgatcctgtt cataatggat gtggcaccca ttacctggac
     1141 ggttgaagta cgtgttgata gtgtagcttg gcatatctgg taacaggcgg ccatagtaag
     1201 ttgagcgtga tccttttccc tcagcataag acttacgaat ttgctccaaa cgcttagacg
     1261 gcacccattc ggggatgtct ttccaatttc ctccgggcgg cacgtgttta atgataagca
     1321 tatgtatatc tccttcttaa agttaaacaa aattatttct agaggggaat tgttatccgc
     1381 tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatcga tctcgatcct
     1441 ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta
     1501 tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg
     1561 tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt
     1621 gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt
     1681 cctaatgcag gagtcgcata agggagagcg tcgagatccc ggacaccatc gaatggcgca
     1741 aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg
     1801 tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt
     1861 cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg
     1921 cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt
     1981 cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg
     2041 cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac
     2101 gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg
     2161 ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca
     2221 ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt
     2281 tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc
     2341 aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct
     2401 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
     2461 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
     2521 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
     2581 ggctgcgcgt tggtgcggac atctcggtag tgggatacga cgataccgaa gacagctcat
     2641 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
     2701 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
     2761 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
     2821 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     2881 gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg
     2941 cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc
     3001 gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc
     3061 tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt
     3121 gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa
     3181 cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc
     3241 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
     3301 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
     3361 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
     3421 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
     3481 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
     3541 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
     3601 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
     3661 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
     3721 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
     3781 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
     3841 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     3901 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     3961 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     4021 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg
     4081 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     4141 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4201 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4261 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4321 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4381 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4441 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4501 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4561 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4621 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4681 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4741 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4801 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4861 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4921 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa
     4981 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa
     5041 cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     5101 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg
     5161 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     5221 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     5281 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc
     5341 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     5401 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     5461 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     5521 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     5581 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     5641 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     5701 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     5761 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     5821 atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5881 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     5941 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     6001 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     6061 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     6121 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     6181 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     6241 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     6301 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     6361 atgcgccgct acagggcgcg tcccattcgc ca
//
