LOCUS       sequence-197159-        4974 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of His-GST-tagged human YTHDC1 YTH domain using 
            mammalian cell-free protein expression system (1-step human 
            high-yield mini IVT kit, cat. #88890, Thermo Fisher Scientific).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4974)
  AUTHORS   Kan L, Grozhik AV, Vedanayagam J, Patil DP, Pang N, Lim KS, Huang 
            YC, Joseph B, Lin CJ, Despic V, Guo J, Yan D, Kondo S, Deng WM, 
            Dedon PC, Jaffrey SR, Lai EC
  TITLE     The m6A pathway facilitates sex determination in Drosophila.
  JOURNAL   Nat Commun. 2017 Jul 4;8:15737. doi: 10.1038/ncomms15737.
  PUBMED    28675155
REFERENCE   2  (bases 1 to 4974)
  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 
            Commun."; date: "2017-07-4"; volume: "8:15737. doi"; pages: " 
            10.1038/ncomms15737"
COMMENT     Sequence Label: pT7CFE1-Nhis-GST-CHA-YTH domain (YTHDC1, 317-532)
FEATURES             Location/Qualifiers
     source          1..4974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     3..22
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3..21
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    72..533
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(128..145)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             553..579
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     CDS             580..1227
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="PILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFE
                     LGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGV
                     SRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYM
                     DPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1185..1207
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1258..1281
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1954..1980
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1954..1980
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2039..2057)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2053..2100
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2129..2146)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2300..2888)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2380..2399)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3059..3850,3851..3919))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3682..3701
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3920..4024)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4092..4110
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4148..4170)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4270..4289
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4358..4813)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4495..4516)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4707..4726
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4939..4961
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4953..4970
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4954..4970
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tgtaatacga ctcactatag ggcgaattaa ttccggttat tttccaccat attgccgtct
       61 tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt
      121 ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct
      181 ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc
      241 ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag
      301 gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc
      361 acctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga
      421 tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaacgtc
      481 taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgatga taatatggcc
      541 accacccata atcaccacca ccaccaccac caccaccacc ctatactagg ttattggaaa
      601 attaagggcc ttgtgcaacc cactcgactt cttttggaat atcttgaaga aaaatatgaa
      661 gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt tgaattgggt
      721 ttggagtttc ccaatcttcc ttattatatt gatggtgatg ttaaattaac acagtctatg
      781 gccatcatac gttatatagc tgacaagcac aacatgttgg gtggttgtcc aaaagagcgt
      841 gcagagattt caatgcttga aggagcggtt ttggatatta gatacggtgt ttcgagaatt
      901 gcatatagta aagactttga aactctcaaa gttgattttc ttagcaagct acctgaaatg
      961 ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt taaatggtga tcatgtaacc
     1021 catcctgact tcatgttgta tgacgctctt gatgttgttt tatacatgga cccaatgtgc
     1081 ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat cccacaaatt
     1141 gataagtact tgaaatccag caagtatata gcatggcctt tgcagggctg gcaagccacg
     1201 tttggtggtg gcgaccatcc tccaaaatcg gatctggttc cgcgtgagcc aaccactctg
     1261 gaagttctgt tccaggggcc cgataaccat atgggatcca gctctgcatc agagtcatat
     1321 gcaggttcag aaaagaagca tgagaaatta tcatcttccg ttcgtgctgt ccgaaaagat
     1381 caaaccagta aactcaaata tgtgcttcaa gatgcaagat ttttcctcat aaagagtaac
     1441 aaccatgaga atgtgtctct tgccaaagcg aagggtgtat ggtccacgct ccctgtaaat
     1501 gagaagaaat taaatcttgc atttagatct gcaaggagtg ttatcttaat attttctgtc
     1561 agagagagtg gaaaatttca agggtttgca agactttctt cagaatcaca tcacggagga
     1621 tctcctatac actgggtgct tccagcagga atgagtgcta aaatgctggg aggtgtcttt
     1681 aaaattgact ggatttgcag gcgtgaatta cccttcacta agtcggctca tctcaccaat
     1741 ccttggaatg aacataaacc agtaaagatc ggacgtgatg gacaggaaat tgaacttgaa
     1801 tgtggaaccc agctttgtct tctgtttccc cccgatgaaa gtattgactt gtatcaggtc
     1861 attcataaaa tgcgtcacaa gagaagaatg cattctcagc cccgatcacg aggacgtcca
     1921 tcccgtcgag aaccagtccg ggatgtgctc gagtacccct acgacgtgcc cgactacgcc
     1981 tgagatctga ctgaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaagtttaa acactagtcc
     2041 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     2101 ctgaaaggag gaactatatc cgggcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2161 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2221 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2281 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2341 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2401 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2461 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2521 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2581 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2641 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2701 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2761 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2821 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2881 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     2941 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3001 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3061 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3121 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3181 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3241 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3301 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3361 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3421 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3481 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3541 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3601 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3661 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3721 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3781 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     3841 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     3901 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     3961 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4021 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     4081 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     4141 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     4201 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     4261 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatatgc ggtgtgaaat
     4321 accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt taatattttg
     4381 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     4441 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     4501 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     4561 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     4621 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     4681 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     4741 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     4801 ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     4861 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     4921 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gaat
//
