LOCUS       sequence-456894-        5738 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression plasmid for YTH domain of YTHDC1 in E.Coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5738)
  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 5738)
  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 YTHDC1
FEATURES             Location/Qualifiers
     source          1..5738
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             716..907
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(901..923)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1009..1148
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1064..1083
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1163..1180)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1334..1922)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1414..1433)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2033..2052)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2044..2859
                     /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             2044..2859
                     /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(2117..2136)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2951..3406)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3088..3109)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3300..3319
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3443..3490)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3486..3504
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4064..4081)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4092..4114
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4129..4153
                     /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(4153..4172)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4154..4172)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4260..4279)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4485..4562
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4563..5645
                     /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     complement(4582..4601)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
       61 cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag
      121 cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc
      181 cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat
      241 ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg
      301 gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg
      361 ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc
      421 gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc
      481 aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg
      541 accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca
      601 acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg
      661 tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac
      721 caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct
      781 tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca
      841 cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa
      901 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
      961 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     1021 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     1081 gtactgagag tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     1141 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     1201 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     1261 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     1321 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     1381 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     1441 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     1501 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     1561 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     1621 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     1681 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     1741 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     1801 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     1861 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     1921 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     1981 aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     2041 gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg
     2101 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     2161 atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     2221 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     2281 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     2341 gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     2401 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     2461 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     2521 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     2581 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     2641 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     2701 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     2761 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     2821 aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata
     2881 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     2941 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     3001 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     3061 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     3121 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     3181 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     3241 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     3301 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     3361 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccaatc
     3421 cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag gccccaaggg
     3481 gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt cgggctttgt
     3541 tagcagccgg atcttagtga cgcattttat gaatgacctg atacaagtca atactttcat
     3601 cggggggaaa cagaagacaa agctgggttc cacattcaag ttcaatttcc tgtccatcac
     3661 gtccgatctt tactggttta tgttcattcc aaggattggt gagatgagcc gacttagtga
     3721 agggtaattc acgcctgcaa atccagtcaa ttttaaagac acctcccagc attttagcac
     3781 tcattcctgc tggaagcacc cagtgtatag gagatcctcc gtgatgtgat tctgaagaaa
     3841 gtcttgcaaa cccttgaaat tttccactct ctctgacaga aaatattaag ataacactcc
     3901 ttgcagatct aaatgcaaga tttaatttct tctcatttac agggagcgtg gaccatacac
     3961 ccttcgcttt ggcaagagac acattctcat ggttgttact ctttatgagg aaaaatcttg
     4021 catcttgaag cacatatttg agtttactgg taccgcttcc accatgatga tgatgatggt
     4081 gcatatgtat atctccttct taaagttaaa caaaattatt tctagagggg aattgttatc
     4141 cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat cgatctcgat
     4201 cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc
     4261 ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc
     4321 ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc
     4381 cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg
     4441 cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc
     4501 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     4561 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     4621 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     4681 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     4741 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     4801 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     4861 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     4921 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     4981 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     5041 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     5101 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     5161 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     5221 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     5281 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     5341 ccgggctgcg cgttggtgcg gacatctcgg tagtgggata cgacgatacc gaagacagct
     5401 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     5461 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     5521 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     5581 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     5641 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg
     5701 atgcccttga gagccttcaa cccagtcagc tccttccg
//
