LOCUS       sequence-439181-        7176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LguI-associated switch methylase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7176)
  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 7176)
  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: POC1481
FEATURES             Location/Qualifiers
     source          1..7176
                     /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"
     CDS             complement(2054..2071)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2081..2098)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2117..2139
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2154..2178
                     /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(2178..2197)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2179..2197)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2281..2300)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2506..2583
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2584..3666
                     /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"
     CDS             4475..4666
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4660..4682)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4768..4907
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4823..4842
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4922..4939)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5093..5681)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5173..5192)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5792..5811)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5803..6618
                     /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             5803..6618
                     /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(5876..5895)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6710..7165)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6847..6868)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7059..7078
                     /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 gcaagctttt
      181 acccggcgcg caaaccatcg cttaacttgt tcgcgatact ctgttgtaaa cgtgcgatct
      241 cgttcaggcg gtcatttaag gctacctctt cgaccagcaa ttcctgcacc tcacgccagt
      301 catcggaagc atccagcact gatgtaggga ccatcagagc cgcaagatca tctgcgtgta
      361 agtgcgccgt ctgaccgcgc accatttcct ggatctgaga ggccactgtc ggttggcgta
      421 agtaggccag cagtaacata ggtgcaatgc gttcttgctt tgggcgcacc aacattactt
      481 ctcccacaaa tgatgcacgg ccgcctaccc attctggaac tttggtgata atgtcaactt
      541 tctttgcaat atagactgga ctgtgtgcgc tactagttaa gacgatgtcg ccgtttttga
      601 ccaattgaat gcccccaagt ttgcgactag tcatcgttga cgtatcaata aagttacggt
      661 cgcgagcaat ccagttaatt ccactcccag tcaagttgcc taccttgata agaaaaagac
      721 cgccattctc actatatgct gcacgaggtg gcgtacgtcc ggtgcgaatc tcgctggcca
      781 aatcacctaa aggaacacca aggttcgagc gacgttttga cgcggacttc gcagacagtg
      841 cctcgaaggt gcggtcagcc ttctggggag ccaggacaga aacataatca cagggtgaac
      901 cttttgggtc gcgcatatgc ttggctaaac catcaagttg cgacccttga cgcgggcgtc
      961 ctgtgctgtc gtatccgaca ttcgataagg tcgcctggac tactcctact ttcttttcac
     1021 ggtccttatc agaagcgtat ttctcaatga aggctaccac tgtgtttgtc tgcgccccgg
     1081 tcaccaagaa cgtttccggt ggcaattgaa tgacagcctt caggtagcca agtttcgcca
     1141 acaagctacg ttcgtacgct gacgatgggt tcgtcagaac gctgcgaggc aacactacag
     1201 ccaaagttcc gccaggcttc agccagcgaa gcgattgttc aacaaataac cattctgagc
     1261 tttggcgggc gcgtggctct ccatttgctg taaacgcgat atcgtacccc gagtaagccg
     1321 cacccgaggc ttcgactgtt acgccgaatg gaggattggt aagaataaaa tcgaagtagc
     1381 cgtagcgaca ccagggttta gaatggcgtt tgcccggttc gacaagcgag tccatcaggg
     1441 cgcgattaaa cgttaagcca gggaagtgac caaggttaag ttctccaatt aaaagcatac
     1501 gagggttctt atccgccccc cagacactga tcttctgcgc attttcctta cgccacttct
     1561 tgatcacttc aattaaaaag gtccccgatc cgcacgcagg atccaggcat ttagcagagc
     1621 tgggcgggtc tacaaacgag actacctcac gtacgacttc atcaggagtc agaaagatcc
     1681 caagtccctt gcgtacttct ggtgtcaaaa actcacgaag agccgccgaa cgaatatcat
     1741 aatcgatctg tgtgaaatta atcccgctga acagagcgtt caattgttct aagcactggt
     1801 ctgaaaggtg aaagtcacga tcgcgccata aactcgtgct ccaactactg tggcgggaca
     1861 ggtggaatgc aaagcgtttg cgaatcgcgc ttcccgttgt aggggcgata ccatcttcct
     1921 cggacatttg ctttaagaat aagaacttca gcagttcctc aaacgcctct tgcggttgaa
     1981 gtccatctaa attgcgcatg gtattgtggg cacgacgata agtactagca agatcacgca
     2041 tgctagccat atggctgccg cgcggcacca ggccgctgct gtgatgatga tgatgatggc
     2101 tgctgcccat ggtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg
     2161 ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc gagatctcga
     2221 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
     2281 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
     2341 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct
     2401 ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
     2461 gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg
     2521 cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     2581 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     2641 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     2701 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     2761 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     2821 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     2881 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     2941 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     3001 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     3061 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     3121 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     3181 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     3241 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     3301 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     3361 tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc
     3421 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     3481 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     3541 cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc
     3601 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     3661 cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc
     3721 gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat
     3781 cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc
     3841 gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc
     3901 gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac
     3961 caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccccac gggtgcgcat
     4021 gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca
     4081 gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac
     4141 ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa
     4201 gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg
     4261 tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg
     4321 gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg
     4381 ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc
     4441 catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct
     4501 taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct
     4561 ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta
     4621 ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     4681 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     4741 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     4801 agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccattgc
     4861 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     4921 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4981 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5041 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5101 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5161 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5221 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5281 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5341 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5401 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5461 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5521 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5581 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5641 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5701 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgaaca
     5761 ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg
     5821 gaaacgtctt gctctaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     5881 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag
     5941 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     6001 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     6061 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca
     6121 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     6181 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     6241 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     6301 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     6361 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     6421 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     6481 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     6541 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     6601 ctcgatgagt ttttctaaga attaattcat gagcggatac atatttgaat gtatttagaa
     6661 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     6721 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     6781 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     6841 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     6901 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     6961 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     7021 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     7081 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     7141 cttaatgcgc cgctacaggg cgcgtcccat tcgcca
//
