LOCUS       sequence-193103-        4977 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 4977)
  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 4977)
  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..4977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    70..531
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(126..143)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             551..577
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     CDS             578..1225
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="PILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFE
                     LGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGV
                     SRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYM
                     DPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1183..1205
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1256..1279
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1952..1978
                     /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             1952..1978
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2036..2054)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2050..2097
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2126..2143)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2297..2885)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2377..2396)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3056..3847,3848..3916))
                     /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     3679..3698
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3917..4021)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4089..4107
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4145..4167)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4267..4286
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4355..4810)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4492..4513)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4704..4723
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4940..4962
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4954..4971
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4955..4971
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 taatacgact cactataggg cgaattaatt ccggttattt tccaccatat tgccgtcttt
       61 tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct
      121 ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct
      181 ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc
      241 acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc
      301 ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctcac
      361 ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc
      421 tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaacgtcta
      481 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac
      541 cacccataat caccaccacc accaccacca ccaccaccct atactaggtt attggaaaat
      601 taagggcctt gtgcaaccca ctcgacttct tttggaatat cttgaagaaa aatatgaaga
      661 gcatttgtat gagcgcgatg aaggtgataa atggcgaaac aaaaagtttg aattgggttt
      721 ggagtttccc aatcttcctt attatattga tggtgatgtt aaattaacac agtctatggc
      781 catcatacgt tatatagctg acaagcacaa catgttgggt ggttgtccaa aagagcgtgc
      841 agagatttca atgcttgaag gagcggtttt ggatattaga tacggtgttt cgagaattgc
      901 atatagtaaa gactttgaaa ctctcaaagt tgattttctt agcaagctac ctgaaatgct
      961 gaaaatgttc gaagatcgtt tatgtcataa aacatattta aatggtgatc atgtaaccca
     1021 tcctgacttc atgttgtatg acgctcttga tgttgtttta tacatggacc caatgtgcct
     1081 ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt gaagctatcc cacaaattga
     1141 taagtacttg aaatccagca agtatatagc atggcctttg cagggctggc aagccacgtt
     1201 tggtggtggc gaccatcctc caaaatcgga tctggttccg cgtgagccaa ccactctgga
     1261 agttctgttc caggggcccg ataaccatat gggatccagc tctgcatcag agtcatatgc
     1321 aggttcagaa aagaagcatg agaaattatc atcttccgtt cgtgctgtcc gaaaagatca
     1381 aaccagtaaa ctcaaatatg tgcttcaaga tgcaagattt ttcctcataa agagtaacaa
     1441 ccatgagaat gtgtctcttg ccaaagcgaa gggtgtatgg tccacgctcc ctgtaaatga
     1501 gaagaaatta aatcttgcat ttagatctgc aaggagtgtt atcttaatat tttctgtcag
     1561 agagagtgga aaatttcaag ggtttgcaag actttcttca gaatcacatc acggaggatc
     1621 tcctatacac tgggtgcttc cagcaggaat gagtgctaaa atgctgggag gtgtctttaa
     1681 aattgactgg atttgcaggc gtgaattacc cttcactaag tcggctcatc tcaccaatcc
     1741 ttggaatgaa cataaaccag taaagatcgg acgtgatgga caggaaattg aacttgaatg
     1801 tggaacccag ctttgtcttc tgtttccccc cgatgaaagt attgacttgt atcaggtcat
     1861 tcataaaatg cgtcacaaga gaagaatgca ttctcagccc cgatcacgag gacgtccatc
     1921 ccgtcgagaa ccagtccggg atgtgctcga gtacccctac gacgtgcccg actacgcctg
     1981 agatctgact gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa agtttaaaca ctagtccgct
     2041 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     2101 aaaggaggaa ctatatccgg gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2161 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2221 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2281 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2341 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2401 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2461 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2521 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2581 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2641 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2701 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     2761 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2821 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2881 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2941 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     3001 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     3061 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3121 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3181 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3241 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3301 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3361 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3421 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3481 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3541 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3601 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3661 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3721 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3781 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3841 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3901 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     3961 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     4021 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     4081 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     4141 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     4201 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     4261 actatgcggc atcagagcag attgtactga gagtgcacca tatatgcggt gtgaaatacc
     4321 gcacagatgc gtaaggagaa aataccgcat caggaaattg taaacgttaa tattttgtta
     4381 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     4441 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     4501 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     4561 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     4621 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     4681 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     4741 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     4801 cagggcgcgt cgcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     4861 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     4921 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattg
//
