LOCUS       sequence-456892-        5756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression plasmid for YTH domain of YTHDF2 in E.Coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5756)
  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 5756)
  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 YTHDF2
FEATURES             Location/Qualifiers
     source          1..5756
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(17..64)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     60..78
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(656..673)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             684..706
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    721..745
                     /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(745..764)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(746..764)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(852..871)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1077..1154
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1155..2237
                     /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(1174..1193)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3046..3237
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3231..3253)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3339..3478
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3394..3413
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3493..3510)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3664..4252)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3744..3763)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4363..4382)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4374..5189
                     /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             4374..5189
                     /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(4447..4466)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5281..5736)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5418..5439)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5630..5649
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tagttcctcc tttcagcaaa aaacccctca agacccgttt agaggcccca aggggttatg
       61 ctagttattg ctcagcggtg gcagcagcca actcagcttc ctttcgggct ttgttagcag
      121 ccggatctta aatggaagtg gtgtgcttgt agctggctat aattttcaac acctgcttag
      181 ccttttccag aggcacttcc tgagtgtccc tagagttggt cactggttta ttctcgttgt
      241 tctctaggcg aatgtgtcgc agttggctat tgggaacgtc cttcacaaaa atccacctga
      301 catcaaaacg acccttccat ttgtcctggg accacacacc tgcacatgtg ttgtagtcca
      361 cagcagattt catttctgcc acgccacaga agtgtccact gccgttgaca ctgaaaagta
      421 agtaaacggg gcctttcccg ttcatggaac gataagcagc atccagtctc ttgttaccat
      481 gctctgtgct gcaccaaata ttatacttaa tggaacggtg aatatcgtcc tcagagtagc
      541 tcttaatgat gaaaacccgg ccatgtttca gattccagtc aaaatctttg gggttatagt
      601 tattaatgga ccgaagcttc tccaacactg ggtggggttc tgaaccgctt ccaccatgat
      661 gatgatgatg gtgcatatgt atatctcctt cttaaagtta aacaaaatta tttctagagg
      721 ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag
      781 atcgatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
      841 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
      901 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
      961 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
     1021 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca
     1081 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     1141 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     1201 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     1261 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     1321 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     1381 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     1441 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     1501 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     1561 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     1621 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     1681 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     1741 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     1801 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     1861 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     1921 ccattaccga gtccgggctg cgcgttggtg cggacatctc ggtagtggga tacgacgata
     1981 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     2041 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     2101 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     2161 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     2221 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg
     2281 ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
     2341 ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag
     2401 gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg
     2461 atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact
     2521 ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggcccca
     2581 cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt
     2641 actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa
     2701 acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg
     2761 gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc
     2821 tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg
     2881 atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt
     2941 aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt
     3001 atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa
     3061 aaaaccgccc ttaacatggc acgctttatc agaagccaga cattaacgct tctggagaaa
     3121 ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct
     3181 gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     3241 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     3301 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     3361 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag
     3421 tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     3481 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3541 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3601 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3661 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3721 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3781 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3841 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3901 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3961 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4021 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4081 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     4141 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4201 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4261 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4321 ggtcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
     4381 atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt
     4441 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat
     4501 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     4561 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     4621 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     4681 ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     4741 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     4801 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     4861 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     4921 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     4981 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     5041 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     5101 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     5161 ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata catatttgaa
     5221 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5281 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     5341 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     5401 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     5461 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     5521 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     5581 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     5641 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     5701 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccaatc cggata
//
