LOCUS       sequence-465600-        6658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Flag-tagged hRPL3 with H245A mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6658)
  AUTHORS   Matsuura-Suzuki E, Shimazu T, Takahashi M, Kotoshiba K, Suzuki T, 
            Kashiwagi K, Sohtome Y, Akakabe M, Sodeoka M, Dohmae N, Ito T, 
            Shinkai Y, Iwasaki S
  TITLE     METTL18-mediated histidine methylation of RPL3 modulates translation
            elongation for proteostasis maintenance.
  JOURNAL   Elife. 2022 Jun 8;11. pii: 72780. doi: 10.7554/eLife.72780.
  PUBMED    35674491
REFERENCE   2  (bases 1 to 6658)
  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: "Elife."; 
            date: "2022-06-8"; volume: "11. pii: 72780. doi"; pages: " 
            10.7554/eLife.72780"
COMMENT     Sequence Label: pcDNA3-hRPL3-FLAG_H245A
FEATURES             Location/Qualifiers
     source          1..6658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2171..2194
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2211..2229)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2212..2229)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2249..2266)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2255..2479
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2525..2953
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2612..2631)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2822..2843
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2962..2982)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2967..3296
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3147..3282
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3209..3228
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3363..4157
                     /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(3417..3436)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4027..4046
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4331..4452
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4368..4387)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4422..4441
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4501..4517)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4501..4517)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4514..4536)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4525..4541
                     /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(4549..4555,4556..4573,4574..4579))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4594..4615
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4732..4749)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4903..5491)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4983..5002)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5662..6453,6454..6522))
                     /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     6285..6304
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6523..6627)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc actattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccactagtaa cggccgccag tgtgctggaa ttcaccatgt ctcacagaaa
      961 gttctccgct cccagacatg ggtccctcgg cttcctgcct cggaagcgca gcagcaggca
     1021 tcgtgggaag gtgaagagct tccctaagga tgacccatcc aagccggtcc acctcacagc
     1081 cttcctggga tacaaggctg gcatgactca catcgtgcgg gaagtcgaca ggccgggatc
     1141 caaggtgaac aagaaggagg tggtggaggc tgtgaccatt gtagagacac cacccatggt
     1201 ggttgtgggc attgtgggct acgtggaaac ccctcgaggc ctccggacct tcaagactgt
     1261 ctttgctgag cacatcagtg atgaatgcaa gaggcgtttc tataagaatt ggcataaatc
     1321 taagaagaag gcctttacca agtactgcaa gaaatggcag gatgaggatg gcaagaagca
     1381 gctggagaag gacttcagca gcatgaagaa gtactgccaa gtcatccgtg tcattgccca
     1441 cacccagatg cgcctgcttc ctctgcgcca gaagaaggcc cacctgatgg agatccaggt
     1501 gaacggaggc actgtggccg agaagctgga ctgggcccgc gagaggcttg agcagcaggt
     1561 acctgtgaac caagtgtttg ggcaggatga gatgatcgac gtcatcgggg tgaccaaggg
     1621 caaaggctac aaaggggtca ccagtcgttg gcacaccaag aagctgcccc gcaagaccgc
     1681 ccgaggcctg cgcaaggtgg cctgtattgg ggcatggcat cctgctcgtg tagccttctc
     1741 tgtggcacgc gctgggcaga aaggctacca tcaccgcact gagatcaaca agaagattta
     1801 taagattggc cagggctacc ttatcaagga cggcaagctg atcaagaaca atgcctccac
     1861 tgactatgac ctatctgaca agagcatcaa ccctctgggt ggctttgtcc actatggtga
     1921 agtgaccaat gactttgtca tgctgaaagg ctgtgtggtg ggaaccaaga agcgggtgct
     1981 caccctccgc aagtccttgc tggtgcagac gaagcggcgg gctctggaga agattgacct
     2041 taagttcatt gacaccacct ccaagtttgg ccatggccgc ttccagacca tggaggagaa
     2101 gaaagcattc atgggaccac tgaagaaaga ccgaattgca aaggaagaag gagcttggcg
     2161 gccgctcgag gactacaagg acgacgatga caagctctag agggccctat tctatagtgt
     2221 cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     2281 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     2341 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     2401 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     2461 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     2521 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2581 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2641 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2701 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2761 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2821 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2881 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2941 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     3001 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     3061 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3121 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     3181 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     3241 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     3301 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     3361 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     3421 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     3481 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     3541 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     3601 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     3661 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     3721 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3781 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3841 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     3901 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     3961 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     4021 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     4081 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     4141 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     4201 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     4261 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     4321 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4381 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4441 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     4501 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4561 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4621 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4681 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4741 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4801 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4861 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4921 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4981 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5041 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5101 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5161 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5221 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5281 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5341 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5401 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5461 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5521 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5581 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5641 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5701 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5761 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5821 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5881 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5941 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6001 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6061 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6121 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6181 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6241 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6301 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6361 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6421 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6481 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6541 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6601 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
