LOCUS       sequence-414390-        6301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing engineered M.EcoGII with higher thermostability.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6301)
  AUTHORS   Ochiai Y, Clifton BE, Le Coz M, Terenzio M, Laurino P
  TITLE     SUPREM: an engineered non-site-specific m6A RNA methyltransferase 
            with highly improved efficiency.
  JOURNAL   Nucleic Acids Res. 2024 Oct 17:gkae887. doi: 10.1093/nar/gkae887.
  PUBMED    39417589
REFERENCE   2  (bases 1 to 6301)
  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: "Nucleic 
            Acids Res. 2024 Oct 17:gkae887. doi: 10.1093/nar/gkae887."
COMMENT     Sequence Label: pET28a-AncMEcoGII250
FEATURES             Location/Qualifiers
     source          1..6301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(559..1374)
                     /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             complement(559..1374)
                     /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     1282..1301
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1366..1385
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1496..2084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1985..2004
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2238..2255
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2270..2409
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2335..2354)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2495..2517
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2511..2702)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3511..4593)
                     /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     4555..4574
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4594..4671)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4877..4896
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4980..4999
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4980..4998
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4999..5023
                     /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)."
     RBS             5038..5060
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6153..6170
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6215..6233)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6229..6276
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      541 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
      601 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
      661 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
      721 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
      781 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
      841 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
      901 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
      961 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     1021 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1081 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     1141 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     1201 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     1261 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     1321 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     1381 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     1441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1621 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     1681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     1921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     1981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2281 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     2341 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     2401 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     2461 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     2521 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     2581 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     2641 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     2701 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     2761 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     2821 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     2881 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     2941 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3001 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3061 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     3121 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     3181 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     3241 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     3301 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     3361 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     3421 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     3481 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3541 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3601 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     3661 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     3721 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     3781 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     3841 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     3901 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     3961 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4021 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4081 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4141 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     4201 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     4261 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     4321 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     4381 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     4441 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     4501 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     4561 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     4621 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     4681 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     4741 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     4801 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     4861 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     4921 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
     4981 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
     5041 gtttaacttt aagaaggaga tataccatga aaaataccgt tcatctgaac tctgtgcagc
     5101 tggtgaacgc ggattctctg cagtacatta aaacgctgcc agataactct gttgatctga
     5161 tcgcgaccga cccgccgtat tttcgtgtga aaagcaacgc ctgggataac cagtgggaat
     5221 ccgaaaccgc tttcctggct tggctggatg aatttctggc ggaattctgg cgtgtactga
     5281 aaccgagcgg ctccctgtac atgttctgcg gcagccgtct ggcggcagat acggaactgc
     5341 tggtacgtca gcgcttcaat gtgctgaacc acattgtttg ggcaaaaccg agcggcccat
     5401 ggcgtcgttg tcgtaaagaa agcctgcgtt ctttctttcc ggcaacggag cgtatcatct
     5461 tcgctgagca ctatggcgca gaaggctacg cgaaaggcca gtctggttac gcagcaaaat
     5521 gtcaggaact gcgccagcag gtgttcaaac cgctgatcga ctacttccgt aacgcgcgtc
     5581 aggccctggg tgtatccgca aaggaaatca accaggccac cggtaagact cagatgtgta
     5641 gccactggtt ctctgagtct cagtggcagc tgccgtctga agaacagtac cagaaactgc
     5701 aggccctgtt tgcccgcaaa gcagctgaac tgggtcaagc cggtgcactg agcaaaagcc
     5761 acaacgagct gactaaagaa tatcaggcac tgagccgcca gtacagcggt ctggtagctc
     5821 agtatgatga cctgaaagcc cagtacgaaa acctgcgtcg tccgttctct gttactaaag
     5881 acgtcccata caccgatgtt tggactttcc cgccggtgca gtactacccg ggtaaacacc
     5941 cgtgtgaaaa accggctgaa atgatggaac acatcatctc tgctagcagc cgtccgggtg
     6001 acgttgttgc ggacttcttc atgggttccg gttctactat caaggcagct ctgaagctgg
     6061 gtcgtcgtgc aatcggtgtg gaactggaag aagaaacctt ccagcaaact gtttctgaaa
     6121 tcgaggatct gggtggtgcg gccgccctcg agcaccacca ccaccaccac tgagatccgg
     6181 ctgctaacaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     6241 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     6301 t
//
