LOCUS       sequence-348690-        7797 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of YTH domain of YTHDC1 tagged with N-terminal 
            mClover3 and C-terminal Mxe GtrA intein-Chitin binding domain for 
            protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7797)
  AUTHORS   Lee JH, Wang R, Xiong F, Krakowiak J, Liao Z, Nguyen PT, Moroz-Omori
            EV, Shao J, Zhu X, Bolt MJ, Wu H, Singh PK, Bi M, Shi CJ, Jamal N, 
            Li G, Mistry R, Jung SY, Tsai KL, Ferreon JC, Stossi F, Caflisch A, 
            Liu Z, Mancini MA, Li W
  TITLE     Enhancer RNA m6A methylation facilitates transcriptional condensate 
            formation and gene activation.
  JOURNAL   Mol Cell. 2021 Aug 19;81(16):3368-3385.e9. doi: 
            10.1016/j.molcel.2021.07.024. Epub 2021 Aug 9.
  PUBMED    34375583
REFERENCE   2  (bases 1 to 7797)
  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: 
            "10.1016/j.molcel.2021.07.024"; journalName: "Mol Cell"; date: 
            "2021-08-19- 19"; volume: "81"; issue: "16"; pages: "3368-3385.e9"
COMMENT     Sequence Label: pTXBI-mClover-YTH domain
FEATURES             Location/Qualifiers
     source          1..7797
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     126..148
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(142..333)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(890..1972)
                     /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     1934..1953
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1973..2050)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      2203..2289
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2386..2472
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2569..2655
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2752..2838
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2935..3021
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3156..3175
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3156..3174
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3175..3199
                     /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)."
     CDS             join(3243..3245,3246..3248,3249..3959)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(3288..3309)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3896..3917
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4386..4979
                     /codon_start=1
                     /gene="Mycobacterium xenopi gyrA"
                     /product="modified GyrA intein (Southworth et al., 1999)"
                     /label=Mxe GyrA intein
                     /note="N-terminal cleavage of this intein can be induced by
                     thiols."
                     /translation="CITGDALVALPEGKSVRIADIVPGARPNSDNAIDLKVLDRHGNPV
                     LADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAV
                     IQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYY
                     AKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA"
     CDS             5010..5165
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     primer_bind     complement(5230..5248)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5244..5291
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        5351..5455
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5456..5524,5525..6316)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5674..5693)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6358..6871)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6553..6574)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6765..6784
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      6982..7570
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7471..7490
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7724..7741
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(7763..7782)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
       61 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
      121 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
      181 gctgcggtaa agctcatcag cgtggtcgtg cagcgattca cagatgtctg cctgttcatc
      241 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
      301 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
      361 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
      421 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
      481 gcgggaccag agaaaaatca ctcagggtca atgccagccg aacgccagca agacgtagcc
      541 cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
      601 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
      661 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
      721 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
      781 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
      841 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
      901 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
      961 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     1021 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     1081 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     1141 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     1201 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     1261 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     1321 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     1381 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     1441 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     1501 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     1561 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     1621 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     1681 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     1741 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     1801 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     1861 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     1921 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     1981 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     2041 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     2101 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg ccggcatgcc
     2161 gccctttcgt cttcaagaat taattcccaa ttccccaggc atcaaataaa acgaaaggct
     2221 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     2281 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
     2341 gcaggacgcc cgccataaac tgccaggaat taattcccca ggcatcaaat aaaacgaaag
     2401 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     2461 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
     2521 cgggcaggac gcccgccata aactgccagg aattaattcc ccaggcatca aataaaacga
     2581 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     2641 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
     2701 tggcgggcag gacgcccgcc ataaactgcc aggaattaat tccccaggca tcaaataaaa
     2761 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     2821 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
     2881 gggtggcggg caggacgccc gccataaact gccaggaatt aattccccag gcatcaaata
     2941 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     3001 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
     3061 ggagggtggc gggcaggacg cccgccataa actgccagga attggggatc ggaattaatt
     3121 cccggtttaa accggggatc tcgatcccgc gaaattaata cgactcacta taggggaatt
     3181 gtgagcggat aacaattccc ctctagaaat aattttgtta actttaagaa ggagatatac
     3241 atatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     3301 acggcgacgt aaacggccac aagttcagcg tccgcggcga gggcgagggc gatgccacca
     3361 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     3421 ccctcgtgac caccttcggc tacggcgtgg cctgcttcag ccgctacccc gaccacatga
     3481 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     3541 ctttcaagga cgacggtacc tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     3601 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     3661 acaagctgga gtacaacttc aacagccaca acgtctatat cacggccgac aagcagaaga
     3721 acggcatcaa ggctaacttc aagatccgcc acaacgttga ggacggcagc gtgcagctcg
     3781 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     3841 actacctgag ccatcagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg
     3901 tcctgctgga gttcgtgacc gccgccggga ttacacatgg catggacgag ctgtacaagt
     3961 atgtgcttca agatgcaaga tttttcctca taaagagtaa caaccatgag aatgtgtctc
     4021 ttgccaaagc gaagggtgta tggtccacgc tccctgtaaa tgagaagaaa ttaaatcttg
     4081 catttagatc tgcaaggagt gttatcttaa tattttctgt cagagagagt ggaaaatttc
     4141 aagggtttgc aagactttct tcagaatcac atcacggagg atctcctata cactgggtgc
     4201 ttccagcagg aatgagtgct aaaatgctgg gaggtgtctt taaaattgac tggatttgca
     4261 ggcgtgaatt acccttcact aagtcggctc atctcaccaa tccttggaat gaacataaac
     4321 cagtaaagat cggacgtgat ggacaggaaa ttgaacttga atgtggaacc cagctttgtc
     4381 ttctgtgcat cacgggagat gcactagttg ccctacccga gggcaagtcg gtacgcatcg
     4441 ccgacatcgt gccgggtgcg cggcccaaca gtgacaacgc catcgacctg aaagtccttg
     4501 accggcatgg caatcccgtg ctcgccgacc ggctgttcca ctccggcgag catccggtgt
     4561 acacggtgcg tacggtcgaa ggtctgcgtg tgacgggcac cgcgaaccac ccgttgttgt
     4621 gtttggtcga cgtcgccggg gtgccgaccc tgctgtggaa gctgatcgac gaaatcaagc
     4681 cgggcgatta cgcggtgatt caacgcagcg cattcagcgt cgactgtgca ggttttgccc
     4741 gcgggaaacc cgaatttgcg cccacaacct acacagtcgg cgtccctgga ctggtgcgtt
     4801 tcttggaagc acaccaccga gacccggacg cccaagctat cgccgacgag ctgaccgacg
     4861 ggcggttcta ctacgcgaaa gtcgccagtg tcaccgacgc cggcgtgcag ccggtgtata
     4921 gccttcgtgt cgacacggca gaccacgcgt ttatcacgaa cgggttcgtc agccacgcta
     4981 ctggcctcac cggtctgaac tcaggcctca cgacaaatcc tggtgtatcc gcttggcagg
     5041 tcaacacagc ttatactgcg ggacaattgg tcacatataa cggcaagacg tataaatgtt
     5101 tgcagcccca cacctccttg gcaggatggg aaccatccaa cgttcctgcc ttgtggcagc
     5161 ttcaatgact gcaggaaggg gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     5221 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     5281 ggggtttttt gctgaaagga ggaactatat ccggataact acgtcaggtg gcacttttcg
     5341 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5401 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5461 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5521 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5581 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5641 acgtttccca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     5701 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5761 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5821 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5881 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5941 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6001 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6061 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6121 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6181 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6241 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6301 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttacc
     6361 ccggttgata atcagaaaag ccccaaaaac aggaagattg tataagcaaa tatttaaatt
     6421 gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     6481 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagcc cgagataggg
     6541 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     6601 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc acccaaatca
     6661 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     6721 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     6781 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     6841 gccgcgctta atgcgccgct acagggcgcg taaaaggatc taggtgaaga tcctttttga
     6901 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6961 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     7021 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     7081 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     7141 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     7201 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     7261 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     7321 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     7381 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7441 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     7501 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     7561 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     7621 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     7681 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     7741 ggaagctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagt
//
