LOCUS       sequence-317531-        7137 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express 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
FEATURES             Location/Qualifiers
     source          1..7137
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        127..506
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        507..710
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     661..681
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     755..774
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        755..773
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             812..835
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2620..2637)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2626..2850
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2896..3324
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2983..3002)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3193..3214
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3333..3353)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3338..3667
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3518..3653
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3580..3599
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3734..4528
                     /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(3788..3807)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4398..4417
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4702..4823
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4739..4758)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4793..4812
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4872..4888)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4872..4888)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4885..4907)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4896..4912
                     /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(4920..4926,4927..4944,4945..4950))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4965..4986
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5103..5120)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5274..5862)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5354..5373)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6033..6824,6825..6893))
                     /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     6656..6675
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6894..6998)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7073..7092)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
       61 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      121 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      181 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      241 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      301 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      361 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      421 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      481 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      541 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      601 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      661 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
      721 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
      781 aagctggcta gcgtttaaac ttaagcttat ggattacaag gatgacgacg ataagtcgga
      841 cacgtggagc tctatccagg cccacaagaa gcagctggac tctctgcggg agaggctgca
      901 gcggaggcgg aagcaggact cggggcactt ggatctacgg aatccagagg cagcattgtc
      961 tccaaccttc cgtagtgaca gcccagtgcc tactgcaccc acctctggtg gccctaagcc
     1021 cagcacagct tcagcagttc ctgaattagc tacagatcct gagttagaga agaagttgct
     1081 acaccacctc tctgatctgg ccttaacatt gcccactgat gctgtgtcca tctgtcttgc
     1141 catctccacg ccagatgctc ctgccactca agatggggta gaaagcctcc tgcagaagtt
     1201 tgcagctcag gagttgattg aggtaaagcg aggtctccta caagatgatg cacatcctac
     1261 tcttgtaacc tatgctgacc attccaagct ctctgccatg atgggtgctg tggcagaaaa
     1321 gaagggccct ggggaggtag cagggactgt cacagggcag aagcggcgtg cagaacagga
     1381 ctcgactaca gtagctgcct ttgccagttc gttagtctct ggtctgaact cttcagcatc
     1441 ggaaccagca aaggagccag ccaagaaatc aaggaaacat gctgcctcag atgttgatct
     1501 ggagatagag agccttctga accaacagtc cactaaggaa caacagagca agaaggtcag
     1561 tcaggagatc ctagagctat taaatactac aacagccaag gaacaatcca ttgttgaaaa
     1621 atttcgctct cgaggtcggg cccaagtgca agaattctgt gactatggaa ccaaggagga
     1681 gtgcatgaaa gccagtgatg ctgatcgacc ctgtcgcaag ctgcacttca gacgaattat
     1741 caataaacac actgatgagt ctttaggtga ctgctctttc cttaatacat gtttccacat
     1801 ggatacctgc aagtatgttc actatgaaat tgatgcttgc atggattctg aggcccctgg
     1861 cagcaaagac cacacgccaa gccaggagct tgctcttaca cagagtgtcg gaggtgattc
     1921 cagtgcagac cgactcttcc cacctcagtg gatctgttgt gatatccgct acctggacgt
     1981 cagtatcttg ggcaagtttg cagttgtgat ggctgaccca ccctgggata ttcacatgga
     2041 actgccctat gggaccctga cagatgatga gatgcgcagg ctcaacatac ccgtactaca
     2101 ggatgatggc tttctcttcc tctgggtcac aggcagggcc atggagttgg ggagagaatg
     2161 tctaaacctc tgggggtatg aacgggtaga tgaaattatt tgggtgaaga caaatcaact
     2221 gcaacgcatc attcggacag gccgtacagg tcactggttg aaccatggga aggaacactg
     2281 cttggttggt gtcaaaggaa atccccaagg cttcaaccag ggtctggatt gtgatgtgat
     2341 cgtagctgag gttcgttcca ccagtcataa accagatgaa atctatggca tgattgaaag
     2401 actatctcct ggcactcgca agattgagtt atttggacga ccacacaatg tgcaacccaa
     2461 ctggatcacc cttggaaacc aactggatgg gatccaccta ctagacccag atgtggttgc
     2521 acggttcaag caaaggtacc cagatggtat catctctaaa cctaagaatt tataggcggc
     2581 cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     2641 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2701 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2761 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     2821 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     2881 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2941 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3001 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3061 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3121 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3181 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3241 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3301 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     3361 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3421 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3481 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     3541 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     3601 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     3661 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     3721 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     3781 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     3841 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     3901 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     3961 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4021 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4081 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4141 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4201 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4261 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     4321 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     4381 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     4441 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4501 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     4561 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4621 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4681 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     4741 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4801 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     4861 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4921 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4981 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5041 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5101 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5161 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5221 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5281 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5341 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5401 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5461 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5521 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5581 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5641 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5701 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5761 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5821 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5881 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5941 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6001 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6061 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6121 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6181 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6241 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6301 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6361 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6421 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6481 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6541 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6601 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6661 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6721 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6781 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6841 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6901 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6961 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7021 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     7081 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcg
//
