LOCUS       sequence-456893-        5777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression plasmid for YTH domain of YTHDF1 in E.Coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5777)
  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 5777)
  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 YTHDF1
FEATURES             Location/Qualifiers
     source          1..5777
                     /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(4103..4120)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4131..4153
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4168..4192
                     /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(4192..4211)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4193..4211)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4299..4318)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4524..4601
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4602..5684
                     /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(4621..4640)
                     /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 atcttattcc ttgcgcacca cctcctcctc ctcctggcgc ttctcgtagt
     3601 gagcaaagtc gtcgaagatg gaggttgtgt gcttgtagga actgataatt ttcagcactt
     3661 gcttggcttt ttctaagggc acctcctggg tgtcccggga gtttgtgacc ggtttgttgt
     3721 cgttattctc cagcctgatg tgccggagct ggttattggg tacatcctta acaaaaatcc
     3781 actggacatc aaacttcccc ttccacttgt cctgagacca gaccccggca ctggtgccgt
     3841 agtccacggg ggacttcatc tcggccaccc cacaaaaatg cccactccca ttgacgctga
     3901 agagcaggta gacgggcccc ttgctgctca tgcagcggaa ggcgctgtcc aggcgcttgt
     3961 tgccgtgctc tgtgctacac cagatggagt acttaatgga gcggtggatg tcgtcctcag
     4021 agtagctctt gatgatgaac acacgcccgc ttttcagatt ccactcaaac tctttcgggt
     4081 tgtagctgtg accgcttcca ccatgatgat gatgatggtg catatgtata tctccttctt
     4141 aaagttaaac aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt
     4201 gagtcgtatt aatttcgcgg gatcgagatc gatctcgatc ctctacgccg gacgcatcgt
     4261 ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga
     4321 tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt
     4381 ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc
     4441 ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca
     4501 taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg
     4561 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     4621 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     4681 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     4741 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     4801 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     4861 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     4921 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     4981 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     5041 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     5101 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     5161 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     5221 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     5281 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     5341 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     5401 acatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     5461 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     5521 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     5581 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     5641 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     5701 gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac
     5761 ccagtcagct ccttccg
//
