LOCUS       sequence-197158-        7056 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of His-tagged human YTHDF3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7056)
  AUTHORS   Patil DP, Chen CK, Pickering BF, Chow A, Jackson C, Guttman M, 
            Jaffrey SR
  TITLE     m6A RNA methylation promotes XIST-mediated transcriptional 
            repression.
  JOURNAL   Nature. 2016 Sep 7;537(7620):369-373. doi: 10.1038/nature19342.
  PUBMED    27602518
REFERENCE   2  (bases 1 to 7056)
  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"; journalName: 
            "Nature"; date: "2016-09-7- 7"; volume: "537"; issue: "7620"; pages:
            "369-373"
COMMENT     Sequence Label: pET-28c(+)-YTHDF3
FEATURES             Location/Qualifiers
     source          1..7056
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      7..462
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(94..113)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     304..325
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(554..1369)
                     /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(554..1369)
                     /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     1277..1296
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1361..1380
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1491..2079
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1980..1999
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2233..2250
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2265..2404
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2330..2349)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2490..2512
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2506..2697)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3506..4588)
                     /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     4550..4569
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4589..4666)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4872..4891
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4975..4994
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4975..4993
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4994..5018
                     /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             5033..5055
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5074..5091
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5101..5118
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             6895..6912
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6965..6983)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6979..7026
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
       61 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
      121 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      181 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
      241 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
      301 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
      361 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
      421 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
      481 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
      541 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
      601 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
      661 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
      721 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
      781 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
      841 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
      901 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
      961 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     1021 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     1081 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     1141 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     1201 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     1261 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     1321 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     1381 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     1441 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     1501 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     1561 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     1621 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     1681 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     1741 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     1801 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     1861 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1921 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1981 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     2041 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     2101 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     2161 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     2221 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     2281 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     2341 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     2401 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     2461 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     2521 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     2581 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     2641 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     2701 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     2761 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     2821 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     2881 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     2941 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     3001 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     3061 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     3121 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     3181 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     3241 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     3301 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     3361 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     3421 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     3481 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     3541 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     3601 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     3661 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     3721 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     3781 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     3841 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     3901 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     3961 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     4021 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     4081 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     4141 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     4201 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     4261 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     4321 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     4381 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     4441 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     4501 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     4561 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     4621 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     4681 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     4741 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     4801 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     4861 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     4921 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
     4981 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     5041 actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca cagcagcggc
     5101 ctggtgccgc gcggcagcca tatggctagc atgtcagcca ctagcgtgga tcagagacct
     5161 aaagggcaag gaaataaagt ttcagtacaa aacggttcga ttcatcaaaa agatgctgta
     5221 aatgatgatg attttgagcc atacttaagt agccagacaa atcagagtaa cagctatcca
     5281 ccaatgtcag atccatacat gcctagttac tatgctccat ccattggatt tccatattct
     5341 cttggggaag cagcgtggtc cacagctgga gaccagccta tgccatatct gacaacctat
     5401 ggacaaatga gtaatggaga acatcactat ataccagatg gtgtatttag tcaacctggg
     5461 gcattaggaa atacccctcc atttcttggt caacatggat ttaacttttt tcctggtaat
     5521 gctgatttct ctacatgggg gacaagtgga tctcagggac aatcaacaca aagttctgct
     5581 tatagtagca gttatggcta tccacctagt tctcttggga gagctattac tgatggacag
     5641 gctggatttg gcaatgatac tttgagtaag gtgcctggca ttagcagtat tgagcaaggc
     5701 atgactggac tgaaaattgg tggtgacctg acagctgcag tgacaaaaac tgtaggtaca
     5761 gctttgagca gcagtggtat gactagcatt gcaaccaata gtgtgccccc agttagcagt
     5821 gcagcaccta aaccaacctc ctgggctgcc attgccagaa agcctgccaa acctcaaccg
     5881 aaacttaaac ccaagggcaa tgtgggaatt gggggttctg ctgtaccacc acctcctata
     5941 aaacacaaca tgaatattgg aacttgggat gaaaaagggt cagtggtaaa ggctccacca
     6001 acccaaccag ttctgcctcc tcaaactata atccagcagc ctcagccatt aattcaacca
     6061 ccaccattgg tgcaaagcca actgcctcaa cagcagcctc aaccaccaca accacagcag
     6121 caacaaggac ctcagccaca ggcccagcct caccaagtgc agcctcaaca gcagcagctg
     6181 cagaatcgct gggtagctcc tcgtaacagg ggagcaggct tcaaccagaa caatggagcg
     6241 ggcagtgaaa actttggttt aggtgttgta cctgtcagtg cttcaccttc tagtgtagaa
     6301 gtgcatcccg tgctggaaaa gctaaaggcc ataaacaact ataatcccaa agactttgat
     6361 tggaatctga agaatggacg tgtgtttata attaaaagct actctgagga tgacatacat
     6421 cgttccatta aatactctat ctggtgtagt actgagcatg gtaataagcg tttggatgca
     6481 gcttaccgtt ccctgaatgg gaaaggccca ctctatttac tcttcagtgt gaatggcagt
     6541 ggacattttt gtggagtggc tgaaatgaag tctgttgtgg actataatgc gtatgctggt
     6601 gtctggtctc aggataagtg gaagggcaaa tttgaagtta aatggatctt tgtcaaagat
     6661 gttcccaata accaattacg gcatattcgc ttagaaaata atgacaacaa accggttacc
     6721 aattcaaggg acactcaaga ggtaccccta gaaaaagcta agcaagtgct taaaataatt
     6781 gctactttca agcataccac ctcaatcttt gatgactttg cacattatga aaagcgtcaa
     6841 gaagaggagg aagccatgcg tagggagaga aatagaaaca aacaataact cgagcaccac
     6901 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     6961 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     7021 tttttgctga aaggaggaac tatatccgga ttggcg
//
