LOCUS       sequence-456896-        5828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression plasmid for YTH domain of YTHDF3 in E.Coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5828)
  AUTHORS   Wang CH, Zhou H
  TITLE     Discovery of a new inhibitor for YTH domain-containing m(6)A RNA 
            readers.
  JOURNAL   RSC Chem Biol. 2024 Jul 25;5(9):914-923. doi: 10.1039/d4cb00105b. 
            eCollection 2024 Aug 28.
  PUBMED    39211476
REFERENCE   2  (bases 1 to 5828)
  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"; doi: 
            "10.1039/d4cb00105b"; journalName: "RSC Chem Biol"; date: 
            "2024-07-25- 25"; volume: "5"; issue: "9"; pages: "914-923"
COMMENT     Sequence Label: PET-28a-YTH domain of YTHDF3
FEATURES             Location/Qualifiers
     source          1..5828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(94..1176)
                     /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     1138..1157
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1177..1254)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1460..1479
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1567..1586
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1567..1585
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1586..1610
                     /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             1625..1647
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1658..1675
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2325..2343)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2339..2386
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2423..2878
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2510..2529)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2720..2741
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2970..3785)
                     /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(2970..3785)
                     /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     3693..3712
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3777..3796
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3907..4495
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4396..4415
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4649..4666
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4681..4820
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4746..4765)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4906..4928
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4922..5113)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
       61 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
      121 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
      181 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
      241 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
      301 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
      361 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
      421 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
      481 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
      541 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
      601 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
      661 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
      721 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
      781 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
      841 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
      901 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
      961 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     1021 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     1081 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     1141 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     1201 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     1261 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     1321 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     1381 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     1441 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     1501 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg
     1561 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     1621 taattttgtt taactttaag aaggagatat acatatgcac catcatcatc atcatggtgg
     1681 aagcggtagt gtagaagtgc atcccgtgct ggaaaagcta aaggccataa acaactataa
     1741 tcccaaagac tttgattgga atctgaagaa tggacgtgtg tttataatta aaagctactc
     1801 tgaggatgac atacatcgtt ccattaaata ctctatctgg tgtagtactg agcatggtaa
     1861 taagcgtttg gatgcagctt accgttccct gaatgggaaa ggcccactct atttactctt
     1921 cagtgtgaat ggcagtggac atttttgtgg agtggctgaa atgaagtctg ttgtggacta
     1981 taatgcgtat gctggtgtct ggtctcagga taagtggaag ggcaaatttg aagttaaatg
     2041 gatctttgtc aaagatgttc ccaataacca attacggcat attcgcttag aaaataatga
     2101 caacaaaccg gttaccaatt caagggacac tcaagaggta cccctagaaa aagctaagca
     2161 agtgcttaaa ataattgcta ctttcaagca taccacctca atctttgatg actttgcaca
     2221 ttatgaaaag cgtcaagaag aggaggaagc catgcgtagg gagagaaata gataagatcc
     2281 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     2341 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     2401 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2461 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2521 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2581 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2641 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2701 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2761 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2821 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     2881 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2941 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
     3001 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     3061 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     3121 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     3181 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     3241 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     3301 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     3361 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     3421 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     3481 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     3541 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     3601 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     3661 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     3721 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     3781 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     3841 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3901 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3961 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4021 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     4081 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4141 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4201 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4261 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4321 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4381 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4441 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4501 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4561 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4621 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4681 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     4741 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     4801 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4861 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4921 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     4981 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     5041 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     5101 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     5161 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     5221 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     5281 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     5341 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     5401 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     5461 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     5521 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     5581 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     5641 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     5701 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     5761 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     5821 gcgcccac
//
