LOCUS       sequence-384008-        6957 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminal MBP fused TadA8.20 in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6957)
  AUTHORS   Xiao YL, Liu S, Ge R, Wu Y, He C, Chen M, Tang W
  TITLE     Transcriptome-wide profiling and quantification of 
            N(6)-methyladenosine by enzyme-assisted adenosine deamination.
  JOURNAL   Nat Biotechnol. 2023 Jan 2. doi: 10.1038/s41587-022-01587-6.
  PUBMED    36593412
REFERENCE   2  (bases 1 to 6957)
  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 
            Biotechnol. 2023 Jan 2. doi: 10.1038/s41587-022-01587-6."
COMMENT     Sequence Label: pET28a-6xHis-MBP-TEV site-TadA8.20
FEATURES             Location/Qualifiers
     source          1..6957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(884..1966)
                     /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     1928..1947
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1967..2044)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2250..2269
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2353..2372
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2353..2371
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2372..2396
                     /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             2411..2433
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2452..2469
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2479..2496
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2500..3600
                     /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"
     primer_bind     3574..3597
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3622..3642
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4174..4191
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4244..4262)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4258..4305
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4342..4797
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4429..4448)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4639..4660
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4889..5704)
                     /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(4889..5704)
                     /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     5612..5631
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5696..5715
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5826..6414
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6315..6334
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6568..6585
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6600..6739
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6665..6684)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6825..6847
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(6841..6957,1..75))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
       61 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
      121 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
      181 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
      241 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
      301 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
      361 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
      421 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
      481 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
      541 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
      601 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
      661 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
      721 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
      781 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
      841 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
      901 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
      961 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     1021 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     1081 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     1141 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     1201 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     1261 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     1321 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     1381 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     1441 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     1501 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     1561 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     1621 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     1681 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     1741 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     1801 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     1861 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     1921 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     1981 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     2041 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     2101 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     2161 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     2221 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     2281 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     2341 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
     2401 tagaaataat tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat
     2461 catcatcaca gcagcggcct ggtgccgcgc ggcagccata tgaaaatcga agaaggtaaa
     2521 ctggtaatct ggattaacgg cgataaaggc tataacggac tcgctgaagt cggtaagaaa
     2581 ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa
     2641 ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc
     2701 tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag
     2761 gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac
     2821 ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca
     2881 aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg
     2941 ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt
     3001 tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct
     3061 ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca
     3121 gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc
     3181 aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta
     3241 ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt
     3301 aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact
     3361 gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct
     3421 tacgaggaag agttggcgaa agatccacgt attgccgcca ctatggaaaa cgcccagaaa
     3481 ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg
     3541 gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact
     3601 tctggaggat ctagcggatc cgaaaacctg tacttccagg gaagcggcag cagcggcggc
     3661 agcatgtctg aggtggagtt ttcccacgag tactggatga gacatgccct gaccctggcc
     3721 aagagggcac gcgatgagag ggaggtgcct gtgggagccg tgctggtgct gaacaataga
     3781 gtgatcggcg agggctggaa cagagccatc ggcctgcacg acccaacagc ccatgccgaa
     3841 attatggccc tgagacaggg cggcctggtc atgcagaact acagactgta tgacgccacc
     3901 ctgtacagca cattcgagcc ttgcgtgatg tgcgccggcg ccatgatcca ctctaggatc
     3961 ggccgcgtgg tgtttggcgt gaggaacgca aaaaccggcg ccgcaggctc cctgatggac
     4021 gtgctgcacc accccggcat gaatcaccgc gtcgaaatta ccgagggaat cctggcagat
     4081 gaatgtgccg ccctgctgtg ccgtttcttt cggatgccta gacgtgtgtt caatgctcag
     4141 aagaaggccc agagctccac cgactaactc gagcatcacc accaccacca ctgagatccg
     4201 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     4261 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     4321 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4381 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4441 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4501 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     4561 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4621 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     4681 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     4741 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     4801 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4861 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     4921 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     4981 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     5041 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     5101 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     5161 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     5221 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     5281 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     5341 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     5401 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     5461 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     5521 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     5581 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     5641 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     5701 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     5761 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5821 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5881 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5941 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     6001 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6061 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6121 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6181 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6241 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6301 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6361 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6421 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     6481 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     6541 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     6601 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     6661 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     6721 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     6781 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     6841 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     6901 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgag
//
