LOCUS       sequence-403596-        7820 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression of full-length YTHDF2 with an N-terminal mCherry 
            tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7820)
  AUTHORS   Dumelie JG, Chen Q, Miller D, Attarwala N, Gross SS, Jaffrey SR
  TITLE     Biomolecular condensates create phospholipid-enriched 
            microenvironments.
  JOURNAL   Nat Chem Biol. 2023 Nov 16. doi: 10.1038/s41589-023-01474-4.
  PUBMED    37973889
REFERENCE   2  (bases 1 to 7820)
  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"; journalName: "Nat Chem 
            Biol. 2023 Nov 16. doi: 10.1038/s41589-023-01474-4."
COMMENT     Sequence Label: pET28C-mCherry-YTHDF2
FEATURES             Location/Qualifiers
     source          1..7820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1857..1874
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1927..1945)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1941..1988
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2025..2480
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2112..2131)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2322..2343
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2572..3387)
                     /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(2572..3387)
                     /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     3295..3314
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3379..3398
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3509..4097
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3998..4017
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4251..4268
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4283..4422
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4348..4367)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4508..4530
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4524..4715)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5524..6606)
                     /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     6568..6587
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6607..6684)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6890..6909
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6993..7012
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6993..7011
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7012..7036
                     /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             7051..7073
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7092..7109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7119..7136
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             7140..7172
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(7179..7820,1..66)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(7324..7342)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7592..7611
                     /label=mCherry-F
                     /note="mCherry, forward primer"
ORIGIN
        1 atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg
       61 tacaagggtg cgcctggttc tgcaggctcc gcagctggcg gatcggggat catgtcggcc
      121 agcagcctct tggagcagag accaaaaggt caaggaaaca aagtacaaaa tggatctgta
      181 catcaaaagg atggattaaa cgatgatgat tttgaacctt acttgagtcc acaggcaagg
      241 cccaataatg catatactgc catgtcagat tcctacttac ccagttacta cagtccctcc
      301 attggcttct cctattcttt gggtgaagct gcttggtcta cggggggtga cacagccatg
      361 ccctacttaa cttcttatgg acagctgagc aacggagagc cccacttcct accagatgca
      421 atgtttgggc aaccaggagc cctaggtagc actccatttc ttggtcagca tggttttaat
      481 ttctttccca gtgggattga cttctcagca tggggaaata acagttctca gggacagtct
      541 actcagagct ctggatatag tagcaattat gcttatgcac ctagctcctt aggtggagcc
      601 atgattgatg gacagtcagc ttttgccaat gagaccctca ataaggctcc tggcatgaat
      661 actatagacc aagggatggc agcactgaag ttgggtagca cagaagttgc aagcaatgtt
      721 ccaaaagttg taggttctgc tgttggtagc gggtccatta ctagtaacat cgtggcttcc
      781 aatagtttgc ctccagccac cattgctcct ccaaaaccag catcttgggc tgatattgct
      841 agcaagcctg caaaacagca acctaaactg aagaccaaga atggcattgc agggtcaagt
      901 cttccgccac ccccgataaa gcataacatg gatattggaa cttgggataa caagggtccc
      961 gttgcaaaag ccccctcaca ggctttggtt cagaatatag gtcagccaac ccaggggtct
     1021 cctcagcctg taggtcagca ggctaacaat agcccaccag tggctcaggc atcagtaggg
     1081 caacagacac agccattgcc tccacctcca ccacagcctg cccagctttc agtccagcaa
     1141 caggcagctc agccaacccg ctgggtagca cctcggaacc gtggcagtgg gttcggtcat
     1201 aatggggtgg atggtaatgg agtaggacag tctcaggctg gttctggatc tactccttca
     1261 gaaccccacc cagtgttgga gaagcttcgg tccattaata actataaccc caaagatttt
     1321 gactggaatc tgaaacatgg ccgggttttc atcattaaga gctactctga ggacgatatt
     1381 caccgttcca ttaagtataa tatttggtgc agcacagagc atggtaacaa gagactggat
     1441 gctgcttatc gttccatgaa cgggaaaggc cccgtttact tacttttcag tgtcaacggc
     1501 agtggacact tctgtggcgt ggcagaaatg aaatctgctg tggactacaa cacatgtgca
     1561 ggtgtgtggt cccaggacaa atggaagggt cgttttgatg tcaggtggat ttttgtgaag
     1621 gacgttccca atagccaact gcgacacatt cgcctagaga acaacgagaa taaaccagtg
     1681 accaactcta gggacactca ggaagtgcct ctggaaaagg ctaagcaggt gttgaaaatt
     1741 atagccagct acaagcacac cacttccatt tttgatgact tctcacacta tgagaaacgc
     1801 caagaggaag aagaaagtgt taaaaaggaa cgtcaaggtc gtgggaaata gtcgagcacc
     1861 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1921 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1981 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     2041 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2101 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2161 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2221 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2281 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2341 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     2401 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     2461 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     2521 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
     2581 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     2641 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     2701 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     2761 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     2821 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     2881 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     2941 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     3001 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     3061 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     3121 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     3181 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     3241 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     3301 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     3361 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     3421 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3481 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3541 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3601 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     3661 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3721 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3781 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3841 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3901 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3961 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4021 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4081 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4141 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4201 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4261 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4321 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4381 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     4441 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4501 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     4561 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     4621 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     4681 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     4741 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     4801 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     4861 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     4921 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     4981 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     5041 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     5101 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     5161 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     5221 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     5281 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     5341 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     5401 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     5461 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     5521 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5581 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     5641 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     5701 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     5761 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     5821 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     5881 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     5941 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     6001 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     6061 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     6121 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     6181 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     6241 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     6301 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     6361 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     6421 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     6481 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     6541 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     6601 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     6661 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     6721 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     6781 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     6841 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     6901 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     6961 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     7021 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca
     7081 tgggcagcag ccatcatcat catcatcaca gcagcggcct ggtgccgcgc ggcagccata
     7141 tggctagcat gactggtgga cagcaaatgg gtcggatcat ggtgagcaag ggcgaggagg
     7201 ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
     7261 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     7321 ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc
     7381 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
     7441 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     7501 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga
     7561 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct
     7621 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc
     7681 agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg
     7741 ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct
     7801 cccacaacga ggactacacc
//
