LOCUS       sequence-392771-        5639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CpG Carboxymethyltransferase (M.MpeI N374K) in bacteria. 
            The gene also encodes an N-terminal (TEV cleavable) MBP tag as well 
            as C-terminal His tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5639)
  AUTHORS   Wang T, Fowler JM, Liu L, Loo CE, Luo M, Schutsky EK, Berrios KN, 
            DeNizio JE, Dvorak A, Downey N, Montermoso S, Pingul BY, Nasrallah 
            M, Gosal WS, Wu H, Kohli RM
  TITLE     Direct enzymatic sequencing of 5-methylcytosine at single-base 
            resolution.
  JOURNAL   Nat Chem Biol. 2023 Jun 15. doi: 10.1038/s41589-023-01318-1.
  PUBMED    37322153
REFERENCE   2  (bases 1 to 5639)
  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: "Nat Chem 
            Biol. 2023 Jun 15. doi: 10.1038/s41589-023-01318-1."
COMMENT     Sequence Label: pMG81-MBP-t-M.MpeI-N374K-His
FEATURES             Location/Qualifiers
     source          1..5639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     204..221
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    236..376
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(302..321)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     462..484
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(604..1230)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     1135..1154
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        1246..1301
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    1252..1270
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    1282..1300
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             1313..2413
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      2270..2279
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2387..2410
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2462..2473
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2486..2506
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3704..3721
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3754..3840
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3932..3959
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3978..4069
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4070..4138,4139..4930)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4288..4307)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5101..5639,1..50)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5590..5609
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
       61 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
      121 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
      181 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
      241 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca
      301 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
      361 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
      421 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
      481 gaggttttca ccgtcatcac cgaaacgcgc gaggcagcag atcaattcgc gcgcgaaggc
      541 gaagcggcat gcgacatctt ggttaccgtg aagttaccat cacggaaaaa ggttatgctg
      601 cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga tcaattcaag
      661 gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag cttgtcgtaa
      721 taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga cttgatgctc
      781 ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg catataatgc
      841 attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga gagtttcata
      901 ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat gacttagtaa
      961 agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt ccccttctaa
     1021 agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt cgagcaaagc
     1081 ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct tctgggcgag
     1141 tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg cgctgttaat
     1201 cactttactt ttatctaatc tagacatcat taattcctaa tttttgttga cactctatca
     1261 ttgatagagt tattttacca ctccctatca gtgatagaga aaagtgcgtg ccatgaaaat
     1321 cgaagaaggt aaactggtaa tctggattaa cggcgataaa ggctataacg ggctcgctga
     1381 agtcggtaag aaattcgaga aagataccgg aattaaagtc accgttgagc atccggataa
     1441 actggaagag aaattcccac aggttgcggc aactggcgat ggccctgaca ttatcttctg
     1501 ggcacacgac cgctttggtg gctacgctca atctggcctg ttggctgaaa tcaccccgga
     1561 caaagcgttc caggacaagc tgtatccgtt tacctgggat gccgtacgtt acaacggcaa
     1621 gctgattgct tacccgatcg ctgttgaagc gttatcgctg atttataaca aagatctgct
     1681 gccgaacccg ccaaaaacct gggaagagat cccggcgctg gataaagaac tgaaagcgaa
     1741 aggtaagagc gcgctgatgt tcaacctgca agaaccgtac ttcacctggc cgctgattgc
     1801 tgctgacggg ggttatgcgt tcaagtatga aaacggcaag tacgacatta aagacgtggg
     1861 cgtggataac gctggcgcga aagcgggtct gaccttcctg gttgacctga ttaaaaacaa
     1921 acacatgaat gcagacaccg attactccat cgcagaagct gcctttaata aaggcgaaac
     1981 agcgatgacc atcaacggcc cgtgggcatg gtccaacatc gacaccagca aagtgaatta
     2041 tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct
     2101 gagcgcaggt attaacgccg ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa
     2161 ctatctgctg actgatgaag gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt
     2221 agcgctgaag tcttacgagg aagagttggc gaaagatcca cgtattgccg ccaccatgga
     2281 aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt tctggtatgc
     2341 cgtgcgtact gcggtgatca acgccgccag cggtcgtcag actgtcgatg aagccctgaa
     2401 agacgcgcag actaattcga gctcgaacaa caacaacaat aacaataaca acaacctcgg
     2461 gatcgaggga aggatttcag aattcgaaaa cctgtatttt caggggtcga caatgaattc
     2521 aaacaaagac aaaattaaag tgattaaggt gtttgaagca tttgcaggta ttggtagtca
     2581 gtttaaggcc ttaaagaata ttgctcgctc aaagaactgg gagatccaac attccgggat
     2641 ggtcgagtgg tttgtggacg cgatcgtatc atacgtcgcc atccatagta aaaactttaa
     2701 cccgaaaatc gaacgccttg atcgtgacat cctcagcatc tcgaatgaca gtaaaatgcc
     2761 catctcagaa tacggaatta aaaaaattaa caatacaatc aaagcgagtt acttgaatta
     2821 tgctaaaaaa cacttcaata acctgtttga catcaagaag gtgaataagg acaatttccc
     2881 gaagaacatc gatatcttta cctattcgtt cccatgtcag gatctgagtg ttcagggcct
     2941 gcagaagggc attgataagg agttgaatac gcgtagcggg cttctgtggg agattgaacg
     3001 gatccttgag gaaattaaga atagtttctc caaagaggaa atgcccaagt atttgttgat
     3061 ggaaaatgtt aagaacttac ttagtcataa aaataaaaaa aactacaaca cctggcttaa
     3121 gcagttagag aagttcggct ataaatccaa aacttatttg cttaattcca agaactttga
     3181 caattgccag aaccgcgaac gcgtgttttg tctttcgatt cgtgacgact acttggagaa
     3241 gacaggattc aagttcaagg agttagagaa ggtcaagaat ccaccgaaaa aaattaagga
     3301 catactagtg gattcatcaa attataaata cctgaatctg aacaagtatg aaacgactac
     3361 tttccgcgaa actaaatcca acatcattag tcgtccactt aaaaactaca ctacgttcaa
     3421 ctccgaaaac tacgtctaca atatcaatgg gatcggacca acactgacag cgagtggcgc
     3481 caactcccgt attaaaattg aaacccaaca gggagtacgt tatttgacgc cattagagtg
     3541 ctttaagtac atgcaattcg atgtaaacga tttcaaaaaa gtgcagtcga caaatttaat
     3601 ttctgagaat aagatgatct acattgcggg gaagtctatc cccgttaaga tcttagaggc
     3661 aatttttaat actttggagt tcgtcaataa tgaggagctt gagcaccatc atcatcatca
     3721 ctaaccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     3781 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     3841 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
     3901 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt
     3961 gcgtttctac aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4021 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     4081 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     4141 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     4201 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     4261 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     4321 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     4381 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     4441 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     4501 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     4561 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     4621 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     4681 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     4741 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtccc gcggtatcat
     4801 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     4861 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     4921 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     4981 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5041 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5101 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5161 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5221 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     5281 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5341 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5401 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5461 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5521 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5581 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgac
//
