LOCUS       sequence-317534-        7137 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express catalytic inactive FLAG-METTL3  in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7137)
  AUTHORS   Sorci M, Ianniello Z, Cruciani S, Larivera S, Ginistrelli LC, 
            Capuano E, Marchioni M, Fazi F, Fatica A
  TITLE     METTL3 regulates WTAP protein homeostasis.
  JOURNAL   Cell Death Dis. 2018 Jul 23;9(8):796. doi: 
            10.1038/s41419-018-0843-z.
  PUBMED    30038300
REFERENCE   2  (bases 1 to 7137)
  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: "Cell Death 
            Dis."; date: "2018-07-23"; volume: "9(8):796. doi"; pages: " 
            10.1038/s41419-018-0843-z"
COMMENT     Sequence Label: pCDNA-FLAG-METTL3-APPA
FEATURES             Location/Qualifiers
     source          1..7137
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        171..550
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        551..754
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     705..725
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     799..818
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        799..817
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             856..879
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2664..2681)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2670..2894
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2940..3368
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3027..3046)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3237..3258
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3377..3397)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3382..3711
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3562..3697
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3624..3643
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3778..4572
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3832..3851)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4442..4461
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4746..4867
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4783..4802)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4837..4856
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4916..4932)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4916..4932)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4929..4951)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4940..4956
                     /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)."
     promoter        complement(join(4964..4970,4971..4988,4989..4994))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5009..5030
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5147..5164)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5318..5906)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5398..5417)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6077..6868,6869..6937))
                     /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     6700..6719
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6938..7042)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7117..7136)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
       61 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
      121 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
      181 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
      241 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      301 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      361 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      421 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      481 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      541 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      601 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      661 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      721 gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc
      781 ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg gctagcgttt
      841 aaacttaagc ttatggatta caaggatgac gacgataagt cggacacgtg gagctctatc
      901 caggcccaca agaagcagct ggactctctg cgggagaggc tgcagcggag gcggaagcag
      961 gactcggggc acttggatct acggaatcca gaggcagcat tgtctccaac cttccgtagt
     1021 gacagcccag tgcctactgc acccacctct ggtggcccta agcccagcac agcttcagca
     1081 gttcctgaat tagctacaga tcctgagtta gagaagaagt tgctacacca cctctctgat
     1141 ctggccttaa cattgcccac tgatgctgtg tccatctgtc ttgccatctc cacgccagat
     1201 gctcctgcca ctcaagatgg ggtagaaagc ctcctgcaga agtttgcagc tcaggagttg
     1261 attgaggtaa agcgaggtct cctacaagat gatgcacatc ctactcttgt aacctatgct
     1321 gaccattcca agctctctgc catgatgggt gctgtggcag aaaagaaggg ccctggggag
     1381 gtagcaggga ctgtcacagg gcagaagcgg cgtgcagaac aggactcgac tacagtagct
     1441 gcctttgcca gttcgttagt ctctggtctg aactcttcag catcggaacc agcaaaggag
     1501 ccagccaaga aatcaaggaa acatgctgcc tcagatgttg atctggagat agagagcctt
     1561 ctgaaccaac agtccactaa ggaacaacag agcaagaagg tcagtcagga gatcctagag
     1621 ctattaaata ctacaacagc caaggaacaa tccattgttg aaaaatttcg ctctcgaggt
     1681 cgggcccaag tgcaagaatt ctgtgactat ggaaccaagg aggagtgcat gaaagccagt
     1741 gatgctgatc gaccctgtcg caagctgcac ttcagacgaa ttatcaataa acacactgat
     1801 gagtctttag gtgactgctc tttccttaat acatgtttcc acatggatac ctgcaagtat
     1861 gttcactatg aaattgatgc ttgcatggat tctgaggccc ctggcagcaa agaccacacg
     1921 ccaagccagg agcttgctct tacacagagt gtcggaggtg attccagtgc agaccgactc
     1981 ttcccacctc agtggatctg ttgtgatatc cgctacctgg acgtcagtat cttgggcaag
     2041 tttgcagttg tgatggctgc cccacccgcc gatattcaca tggaactgcc ctatgggacc
     2101 ctgacagatg atgagatgcg caggctcaac atacccgtac tacaggatga tggctttctc
     2161 ttcctctggg tcacaggcag ggccatggag ttggggagag aatgtctaaa cctctggggg
     2221 tatgaacggg tagatgaaat tatttgggtg aagacaaatc aactgcaacg catcattcgg
     2281 acaggccgta caggtcactg gttgaaccat gggaaggaac actgcttggt tggtgtcaaa
     2341 ggaaatcccc aaggcttcaa ccagggtctg gattgtgatg tgatcgtagc tgaggttcgt
     2401 tccaccagtc ataaaccaga tgaaatctat ggcatgattg aaagactatc tcctggcact
     2461 cgcaagattg agttatttgg acgaccacac aatgtgcaac ccaactggat cacccttgga
     2521 aaccaactgg atgggatcca cctactagac ccagatgtgg ttgcacggtt caagcaaagg
     2581 tacccagatg gtatcatctc taaacctaag aatttatagg cggccgctcg agtctagagg
     2641 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2701 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2761 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2821 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2881 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     2941 cgcgccctgt agcggcgcat taagcgcgac gggtgtggtg gttacgcgca gcgtgaccgc
     3001 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3061 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3121 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3181 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3241 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3301 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3361 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3421 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3481 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3541 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3601 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     3661 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     3721 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     3781 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     3841 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     3901 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     3961 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     4021 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     4081 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     4141 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     4201 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     4261 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     4321 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4381 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     4441 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4501 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     4561 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     4621 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     4681 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4741 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4801 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4861 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     4921 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4981 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5041 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5101 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5161 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5221 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5281 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5341 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5401 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5461 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5521 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5581 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5641 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5701 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5761 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5821 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5881 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5941 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6001 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6061 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6121 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6181 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6241 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6301 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6361 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6421 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6481 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6541 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6601 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6661 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6721 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6781 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6841 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6901 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6961 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7021 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     7081 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgc
//
