LOCUS       sequence-456895-        5663 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression plasmid for YTH domain of YTHDC2 in E.Coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5663)
  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 5663)
  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 YTHDC2
FEATURES             Location/Qualifiers
     source          1..5663
                     /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(2160..2178)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2174..2221
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2258..2713
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2345..2364)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2555..2576
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2805..3620)
                     /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(2805..3620)
                     /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     3528..3547
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3612..3631
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3742..4330
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4231..4250
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4484..4501
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4516..4655
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4581..4600)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4741..4763
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4757..4948)
                     /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 aagcggtaac atgcctgttc gatacttcat aatgaagagt agcaatttga gaaaccttga
     1741 aatttctcaa cagaagggta tctggtctac aactcctagt aatgaacgga agctaaatcg
     1801 agccttttgg gaaagcagca tagtttactt ggtattttct gttcaaggat ctggacattt
     1861 ccagggattt tctaggatgt cttctgagat tggaagggaa aagagtcagg actggggctc
     1921 tgctggacta ggaggagtat ttaaggtgga gtggatacga aaagaaagcc ttccctttca
     1981 atttgcacac catttactca atccatggaa tgacaacaag aaagtgcaga taagcaggga
     2041 tgggcaggaa ctagaacctc tggttggtga acagttgctc cagttatggg aacgtcttcc
     2101 cttgggataa gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     2161 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     2221 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     2281 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2341 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2401 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2461 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2521 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2581 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2641 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     2701 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2761 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
     2821 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     2881 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     2941 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     3001 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     3061 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     3121 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     3181 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     3241 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     3301 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     3361 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     3421 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     3481 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     3541 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     3601 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     3661 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3721 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3781 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3841 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3901 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3961 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4021 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4081 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4141 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4201 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4261 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4321 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4381 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4441 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4501 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4561 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     4621 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     4681 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4741 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     4801 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     4861 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     4921 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     4981 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     5041 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     5101 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     5161 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     5221 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     5281 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     5341 tctgctaacc agtaaggcaa ccacgccagc ctagccgggt cctcaacgac aggagcacga
     5401 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     5461 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     5521 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     5581 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     5641 cgatagtcat gccccgcgcc cac
//
