LOCUS       sequence-335119-        7814 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. cerevisiae (yeast) vector for constitutive expression of 
            3xFLAG-Nop4 WT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7814)
  AUTHORS   Bryant CJ, Lorea CF, de Almeida HL Jr, Weinert L, Vedolin L, Pinto E
            Vairo F, Baserga SJ
  TITLE     Biallelic splicing variants in the nucleolar 60S assembly factor 
            RBM28 cause the ribosomopathy ANE syndrome.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 May 11;118(19). pii: 2017777118. doi:
            10.1073/pnas.2017777118.
  PUBMED    33941690
REFERENCE   2  (bases 1 to 7814)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2021-05-11"; volume: "118(19). pii: 
            2017777118. doi"; pages: " 10.1073/pnas.2017777118"
COMMENT     Sequence Label: p414GPD-3xFLAG-Nop4 WT
FEATURES             Location/Qualifiers
     source          1..7814
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     67..89
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(127..145)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    186..689
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        726..830
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(831..899,900..1691)
                     /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     complement(1049..1068)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1862..2450
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2351..2370
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2604..2621
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2738..2759
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2774..2779,2780..2797,2798..2804)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2812..2828
                     /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)."
     primer_bind     2817..2839
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2836..2852
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2836..2852
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2871..2891
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2873..2891
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2916..3559
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3453..3476
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     3565..3581
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3565..3581
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             3598..3663
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    3664..3688
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5750..5774)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5777..6024
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5793..5811)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(6042..6061)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6043..6061)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6071..6088)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6071..6087)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6080..6102)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6228..6683
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6315..6334)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6525..6546
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6786..7460)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(7461..7741)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(7762..7781)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
       61 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
      121 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
      181 agacggatcg cttgcctgta acttacacgc gcctcgtatc ttttaatgat ggaataattt
      241 gggaatttac tctgtgttta tttattttta tgttttgtat ttggatttta gaaagtaaat
      301 aaagaaggta gaagagttac ggaatgaaga aaaaaaaata aacaaaggtt taaaaaattt
      361 caacaaaaag cgtactttac atatatattt attagacaag aaaagcagat taaatagata
      421 tacattcgat taacgataag taaaatgtaa aatcacagga ttttcgtgtg tggtcttcta
      481 cacagacaag atgaaacaat tcggcattaa tacctgagag caggaagagc aagataaaag
      541 gtagtatttg ttggcgatcc ccctagagtc ttttacatct tcggaaaaca aaaactattt
      601 tttctttaat ttcttttttt actttctatt tttaatttat atatttatat taaaaaattt
      661 aaattataat tatttttata gcacgtgatg aaaaggaccc aggtggcact tttcggggaa
      721 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
      781 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
      841 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
      901 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
      961 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     1021 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     1081 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     1141 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     1201 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     1261 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     1321 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     1381 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     1441 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     1501 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     1561 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     1621 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     1681 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     1741 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     1801 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     1861 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     1921 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     1981 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     2041 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     2101 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     2161 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     2221 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     2281 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     2341 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     2401 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     2461 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     2521 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     2581 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     2641 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     2701 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     2761 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     2821 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc
     2881 ctcactaaag ggaacaaaag ctggagctca gtttatcatt atcaatactc gccatttcaa
     2941 agaatacgta aataattaat agtagtgatt ttcctaactt tatttagtca aaaaattagc
     3001 cttttaattc tgctgtaacc cgtacatgcc caaaataggg ggcgggttac acagaatata
     3061 taacatcgta ggtgtctggg tgaacagttt attcctggca tccactaaat ataatggagc
     3121 ccgcttttta agctggcatc cagaaaaaaa aagaatccca gcaccaaaat attgttttct
     3181 tcaccaacca tcagttcata ggtccattct cttagcgcaa ctacagagaa caggggcaca
     3241 aacaggcaaa aaacgggcac aacctcaatg gagtgatgca acctgcctgg agtaaatgat
     3301 gacacaaggc aattgaccca cgcatgtatc tatctcattt tcttacacct tctattacct
     3361 tctgctctct ctgatttgga aaaagctgaa aaaaaaggtt gaaaccagtt ccctgaaatt
     3421 attcccctac ttgactaata agtatataaa gacggtaggt attgattgta attctgtaaa
     3481 tctatttctt aaacttctta aattctactt ttatagttag tctttttttt agttttaaaa
     3541 caccagaact tagtttcgac ggattctaga actagtggat cccccgggct gcagatggac
     3601 tacaaagacc atgacggtga ttataaagat catgatatcg attacaagga tgacgatgac
     3661 aagacaagtt tgtacaaaaa agcaggctac aaaatggaag aaacaataga aaatgttgaa
     3721 gtgccttcta gtaatgtctc aaaacaaaat gacgacggcc tagatatgaa gactttgttt
     3781 gtgaggtcta taccccagga tgtcacagat gagcagctgg cagatttctt ttccaatttt
     3841 gctccgatca agcatgcggt agttgttaag gacacgaata aaaggtccag gggcttcggg
     3901 ttcgttagtt ttgctgtaga ggatgacaca aaagaagccc tagctaaggc cagaaagacc
     3961 aaatttaacg gacatatctt gagggtagac attgctaaaa gaagagatcg ttcaaagaaa
     4021 acatcagaag tggttgaaaa gagcacacct gagtcatctg agaaaataac gggccagaac
     4081 aatgaagatg aggacgatgc tgatggtgaa gactccatgc taaaaggtaa accgaaatta
     4141 attatcagaa atatgccatg gagttgtcgt gatccagtta aattgaaaaa gatctttggt
     4201 agatatggta ccgtcgtgga ggctaccatt ccaaggaagc gtgatggtaa attgtgtgga
     4261 tttgcgtttg ttactatgaa gaaaatcagt aattgtagaa ttgctttgga aaacaccaaa
     4321 gacttgaaaa ttgatggtag aaaggtggct gttgattttg ctgttcaaaa aaacagatgg
     4381 gaagattaca aaaaagccca gccagaaatg aatgacaaag atgataacga gtcaggaaat
     4441 gaagatgctg aagaaaacca tgacgacgaa gaagatgaaa atgaagagga agatagacaa
     4501 gtagatcaag cttcaaaaaa caaagaatcg aaacgtaaag cacaaaacaa aagggaagat
     4561 ttttctgttt ttgttcgtaa cgtcccttat gatgccactg aagaatcttt ggctccgcac
     4621 ttcagtaaat ttggcagcgt taaatacgca ttacctgtta tcgataagtc tacggggttg
     4681 gctaaaggta cggcctttgt agccttcaaa gaccaatata catataacga gtgtatcaaa
     4741 aatgcgcctg cggctggctc tacctcttta ttgataggtg atgatgtcat gcctgagtat
     4801 gtgtatgaag gtcgtgttct gtccattact ccaacgttag taagagagga tgctggaaga
     4861 atggcagaaa agaacgccgc caaaagaaag gaggccctag gtaaagcgcc aggcgaaaaa
     4921 gatagacgta acttatattt gctgaatgaa ggtagagttg ttgaaggctc aaaaatggct
     4981 gatttgttaa ccaacactga catggagatc agagaaaaat cttacaaatt aagagtggag
     5041 caactgaaaa agaacccatc tttgcatttg tcgatgacta ggttggctat cagaaatctt
     5101 cctagagcaa tgaatgataa agcattaaag gcgttagctc gtaaagctgt cgtggaattt
     5161 gctacagaag ttaaaaataa ggaaagacat ccattgagta aagaagagat tatcagatct
     5221 acaaaggaaa aatacaagtt catgggtcca gatgagattg aagctcaaaa aaagaaagac
     5281 aaaaaaagtg gtgttgtcaa gcaagcaaaa gttataatgg aagttaaagg atcaactgcg
     5341 ggaagaagta gaggttacgg ttttgtagaa ttcagagatc ataaaaacgc tttgatgggc
     5401 ttaaggtggt tgaattgtca tgccgttacc tccgatgaaa ttttagaggg tttgaatgat
     5461 gatgaaaaga aacaagtaga caatgactta ggcaaaggaa gacgtctttg tgttgaattt
     5521 gcgattgaga actctaacgt cgtaaagaga agaagggaac aattgaagca agcaagaacc
     5581 aagagaacaa gaccagataa tgaagatact ggagatgtcg gagaatccga gaataagaag
     5641 ccaaaaaaag aagaagcaac gacgcccacc aatcccgatg acaaaaagat gggcgatgat
     5701 ataaagagaa taatcgggtt caagcgtaag agaaagcacg caaaaaaaaa cccagctttc
     5761 ttgtacaaag tggtagtcat gtaattagtt atgtcacgct tacattcacg ccctcccccc
     5821 acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt ccctatttat
     5881 ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt
     5941 ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg
     6001 ggacgctcga aggctttaat ttgcggccgg tacccaattc gccctatagt gagtcgtatt
     6061 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     6121 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     6181 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggacg cgccctgtag
     6241 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     6301 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     6361 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     6421 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     6481 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     6541 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     6601 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     6661 caaaatatta acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt
     6721 atttcacacc gcatagatcg gcaagtgcac aaacaatact taaataaata ctactcagta
     6781 ataacctatt tcttagcatt tttgacgaaa tttgctattt tgttagagtc ttttacacca
     6841 tttgtctcca cacctccgct tacatcaaca ccaataacgc catttaatct aagcgcatca
     6901 ccaacatttt ctggcgtcag tccaccagct aacataaaat gtaagctttc ggggctctct
     6961 tgccttccaa cccagtcaga aatcgagttc caatccaaaa gttcacctgt cccacctgct
     7021 tctgaatcaa acaagggaat aaacgaatga ggtttctgtg aagctgcact gagtagtatg
     7081 ttgcagtctt ttggaaatac gagtctttta ataactggca aaccgaggaa ctcttggtat
     7141 tcttgccacg actcatctcc atgcagttgg acgatatcaa tgccgtaatc attgaccaga
     7201 gccaaaacat cctccttagg ttgattacga aacacgccaa ccaagtattt cggagtgcct
     7261 gaactatttt tatatgcttt tacaagactt gaaattttcc ttgcaataac cgggtcaatt
     7321 gttctctttc tattgggcac acatataata cccagcaagt cagcatcgga atctagagca
     7381 cattctgcgg cctctgtgct ctgcaagccg caaactttca ccaatggacc agaactacct
     7441 gtgaaattaa taacagacat actccaagct gcctttgtgt gcttaatcac gtatactcac
     7501 gtgctcaata gtcaccaatg ccctccctct tggccctctc cttttctttt ttcgaccgaa
     7561 ttaattctta atcggcaaaa aaagaaaagc tccggatcaa gattgtacgt aaggtgacaa
     7621 gctatttttc aataaagaat atcttccact actgccatct ggcgtcataa ctgcaaagta
     7681 cacatatatt acgatgctgt tctattaaat gcttcctata ttatatatat agtaatgtcg
     7741 tgatctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     7801 acacccgcca acac
//
