LOCUS       sequence-216987-        6932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminally FLAG-tagged human RNMT in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6932)
  AUTHORS   Trotman JB, Giltmier AJ, Mukherjee C, Schoenberg DR
  TITLE     RNA guanine-7 methyltransferase catalyzes the methylation of 
            cytoplasmically recapped RNAs.
  JOURNAL   Nucleic Acids Res. 2017 Oct 13;45(18):10726-10739. doi: 
            10.1093/nar/gkx801.
  PUBMED    28981715
REFERENCE   2  (bases 1 to 6932)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2017-10-13- 13"; volume: "45"; issue: 
            "18"; pages: "10726-10739"
COMMENT     Sequence Label: pcDNA3-FLAG-RNMT
FEATURES             Location/Qualifiers
     source          1..6932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        27..406
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        407..610
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     561..581
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     655..674
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        655..673
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             694..717
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             730..741
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        complement(2277..2295)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2278..2295)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2315..2332)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2321..2545
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2591..3019
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2678..2697)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2888..2909
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3028..3048)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3033..3362
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3213..3348
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3275..3294
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3429..4223
                     /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(3483..3502)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4093..4112
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4397..4518
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4434..4453)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4488..4507
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4567..4583)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4567..4583)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4580..4602)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4591..4607
                     /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(4615..4621,4622..4639,4640..4645))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4660..4681
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4798..4815)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4969..5557)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5049..5068)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5728..6519,6520..6588))
                     /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     6351..6370
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6589..6693)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6768..6787)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
       61 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      121 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      181 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      241 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      301 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      361 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      421 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      481 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      541 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      601 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac
      661 gactcactat agggagaccc aagcttgatg ggcgactaca aagacgatga cgacaagagc
      721 agcggccata tcgaaggtcg tcatatggca aattctgcaa aagcagaaga atatgaaaag
      781 atgtctcttg aacaggcaaa agcgtcagtg aattctgaaa cagagtcttc attcaatatt
      841 aatgaaaaca caacagcttc tgggactggg ctttctgaaa agacttctgt ctgtaggcaa
      901 gtagacatag caagaaagag aaaagagttt gaagatgatc ttgtaaagga aagttctagt
      961 tgtgggaaag acactccatc caagaagaga aaacttgatc ctgaaattgt cccagaggaa
     1021 aaagattgtg gtgatgctga aggcaattca aagaaaagaa aaagagaaac tgaggatgtt
     1081 ccaaaagata aatcttctac tggagatggc actcaaaata agagaaaaat agcacttgag
     1141 gatgttcctg aaaagcagaa aaatctggaa gaaggacaca gctcaacagt ggctgcccat
     1201 tacaatgaac ttcaggaagt tggtttggag aagcgtagtc aaagtcgtat tttttaccta
     1261 agaaacttta ataattggat gaaaagtgtt ctcattggag aatttttgga aaaggtacga
     1321 cagaagaaaa aacgtgatat cactgttttg gacctgggat gtggtaaagg tggagatttg
     1381 ctgaaatgga aaaaaggaag aattaacaag ctagtttgta ctgatattgc cgatgtttct
     1441 gtcaaacagt gtcagcagcg gtatgaggac atgaaaaatc gtcgtgatag tgaatatatt
     1501 ttcagtgcag aatttataac tgctgacagc tcaaaggaac ttctgattga caaatttcgt
     1561 gacccacaaa tgtgttttga catctgcagt tgtcagtttg tctgtcatta ctcatttgag
     1621 tcttatgagc aggctgacat gatgctgaga aatgcgtgtg agagacttag ccctgggggc
     1681 tattttattg gtactactcc caatagcttt gaattgataa gacgccttga agcttcagaa
     1741 acagaatcat ttggaaatga aatatatact gtgaaatttc agaagaaagg agattatcct
     1801 ttatttggct gcaaatatga cttcaacttg gaaggtgttg tggatgttcc tgaattcttg
     1861 gtctattttc cattgctaaa tgaaatggca aagaagtaca atatgaaact agtctacaaa
     1921 aaaacatttc tggaattcta cgaagaaaag attaagaaca atgaaaataa aatgctctta
     1981 aaacgaatgc aggccttgga gccatatcct gcaaatgaga gttctaaact tgtctctgag
     2041 aaggtggatg actatgaaca tgcagcaaag tacatgaaga acagtcaagt aaggttacct
     2101 ttgggaacct taagtaaatc agaatgggaa gctacaagta tttacttggt gtttgccttt
     2161 gagaaacagc agtgatgacg agctcggatc cactagtaac ggccgccagt gtgctggaat
     2221 tctgcagata tccatcacac tggcggccgc tcgagcatgc atctagaggg ccctattcta
     2281 tagtgtcacc taaatgctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     2341 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     2401 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     2461 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     2521 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     2581 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2641 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2701 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2761 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2821 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2881 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     2941 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3001 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3061 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3121 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3181 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3241 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3301 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3361 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     3421 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     3481 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     3541 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     3601 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     3661 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     3721 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     3781 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     3841 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     3901 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     3961 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4021 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4081 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     4141 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4201 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4261 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4321 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4381 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4441 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4501 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     4561 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4621 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4681 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4741 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4801 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4861 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4921 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4981 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5041 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5101 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5161 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5221 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5281 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5341 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5401 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5461 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5521 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5581 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5641 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5701 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5761 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5821 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5881 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5941 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6001 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6061 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6121 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6181 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6241 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6301 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6361 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6421 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6481 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6541 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6601 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6661 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6721 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     6781 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     6841 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     6901 ctgcttaggg ttaggcgttt tgcgctgctt cg
//
