LOCUS       sequence-335124-        7721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. cerevisiae (yeast) vector for constitutive expression of 
            3xFLAG-Nop4 delta Exon 5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7721)
  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 7721)
  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 dE5
FEATURES             Location/Qualifiers
     source          1..7721
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     3..20
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    137..158
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(173..178,179..196,197..203)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    211..227
                     /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     216..238
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     235..251
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     235..251
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     270..290
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        272..290
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        315..958
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     852..875
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     964..980
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     964..980
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             997..1062
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    1063..1087
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3056..3080)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      3083..3330
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3099..3117)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3348..3367)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3349..3367)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3377..3394)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3377..3393)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3386..3408)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3534..3989
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3621..3640)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3831..3852
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4092..4766)
                     /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(4767..5047)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(5068..5087)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5187..5209
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5247..5265)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5306..5809
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5846..5950
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5951..6019,6020..6811)
                     /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(6169..6188)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6982..7570
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7471..7490
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
       61 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
      121 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
      181 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
      241 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
      301 tggagctcag tttatcatta tcaatactcg ccatttcaaa gaatacgtaa ataattaata
      361 gtagtgattt tcctaacttt atttagtcaa aaaattagcc ttttaattct gctgtaaccc
      421 gtacatgccc aaaatagggg gcgggttaca cagaatatat aacatcgtag gtgtctgggt
      481 gaacagttta ttcctggcat ccactaaata taatggagcc cgctttttaa gctggcatcc
      541 agaaaaaaaa agaatcccag caccaaaata ttgttttctt caccaaccat cagttcatag
      601 gtccattctc ttagcgcaac tacagagaac aggggcacaa acaggcaaaa aacgggcaca
      661 acctcaatgg agtgatgcaa cctgcctgga gtaaatgatg acacaaggca attgacccac
      721 gcatgtatct atctcatttt cttacacctt ctattacctt ctgctctctc tgatttggaa
      781 aaagctgaaa aaaaaggttg aaaccagttc cctgaaatta ttcccctact tgactaataa
      841 gtatataaag acggtaggta ttgattgtaa ttctgtaaat ctatttctta aacttcttaa
      901 attctacttt tatagttagt ctttttttta gttttaaaac accagaactt agtttcgacg
      961 gattctagaa ctagtggatc ccccgggctg cagatggact acaaagacca tgacggtgat
     1021 tataaagatc atgatatcga ttacaaggat gacgatgaca agacaagttt gtacaaaaaa
     1081 gcaggctaca aaatggaaga aacaatagaa aatgttgaag tgccttctag taatgtctca
     1141 aaacaaaatg acgacggcct agatatgaag actttgtttg tgaggtctat accccaggat
     1201 gtcacagatg agcagctggc agatttcttt tccaattttg ctccgatcaa gcatgcggta
     1261 gttgttaagg acacgaataa aaggtccagg ggcttcgggt tcgttagttt tgctgtagag
     1321 gatgacacaa aagaagccct agctaaggcc agaaagacca aatttaacgg acatatcttg
     1381 agggtagaca ttgctaaaag aagagatcgt tcaaagaaaa catcagaagt ggttgaaaag
     1441 agcacacctg agtcatctga gaaaataacg ggccagaaca atgaagatga ggacgatgct
     1501 gatggtgaag actccatgct aaaaggtaaa ccgaaattaa ttatcagaaa tatgccatgg
     1561 agttgtcgtg atccagttaa attgaaaaag atctttggta gatatggtac cgtcgtggag
     1621 gctaccattc caaggaagcg tggtagaaag gtggctgttg attttgctgt tcaaaaaaac
     1681 agatgggaag attacaaaaa agcccagcca gaaatgaatg acaaagatga taacgagtca
     1741 ggaaatgaag atgctgaaga aaaccatgac gacgaagaag atgaaaatga agaggaagat
     1801 agacaagtag atcaagcttc aaaaaacaaa gaatcgaaac gtaaagcaca aaacaaaagg
     1861 gaagattttt ctgtttttgt tcgtaacgtc ccttatgatg ccactgaaga atctttggct
     1921 ccgcacttca gtaaatttgg cagcgttaaa tacgcattac ctgttatcga taagtctacg
     1981 gggttggcta aaggtacggc ctttgtagcc ttcaaagacc aatatacata taacgagtgt
     2041 atcaaaaatg cgcctgcggc tggctctacc tctttattga taggtgatga tgtcatgcct
     2101 gagtatgtgt atgaaggtcg tgttctgtcc attactccaa cgttagtaag agaggatgct
     2161 ggaagaatgg cagaaaagaa cgccgccaaa agaaaggagg ccctaggtaa agcgccaggc
     2221 gaaaaagata gacgtaactt atatttgctg aatgaaggta gagttgttga aggctcaaaa
     2281 atggctgatt tgttaaccaa cactgacatg gagatcagag aaaaatctta caaattaaga
     2341 gtggagcaac tgaaaaagaa cccatctttg catttgtcga tgactaggtt ggctatcaga
     2401 aatcttccta gagcaatgaa tgataaagca ttaaaggcgt tagctcgtaa agctgtcgtg
     2461 gaatttgcta cagaagttaa aaataaggaa agacatccat tgagtaaaga agagattatc
     2521 agatctacaa aggaaaaata caagttcatg ggtccagatg agattgaagc tcaaaaaaag
     2581 aaagacaaaa aaagtggtgt tgtcaagcaa gcaaaagtta taatggaagt taaaggatca
     2641 actgcgggaa gaagtagagg ttacggtttt gtagaattca gagatcataa aaacgctttg
     2701 atgggcttaa ggtggttgaa ttgtcatgcc gttacctccg atgaaatttt agagggtttg
     2761 aatgatgatg aaaagaaaca agtagacaat gacttaggca aaggaagacg tctttgtgtt
     2821 gaatttgcga ttgagaactc taacgtcgta aagagaagaa gggaacaatt gaagcaagca
     2881 agaaccaaga gaacaagacc agataatgaa gatactggag atgtcggaga atccgagaat
     2941 aagaagccaa aaaaagaaga agcaacgacg cccaccaatc ccgatgacaa aaagatgggc
     3001 gatgatataa agagaataat cgggttcaag cgtaagagaa agcacgcaaa aaaaaaccca
     3061 gctttcttgt acaaagtggt agtcatgtaa ttagttatgt cacgcttaca ttcacgccct
     3121 ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct
     3181 atttattttt ttatagttat gttagtatta agaacgttat ttatatttca aatttttctt
     3241 ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag
     3301 gttttgggac gctcgaaggc tttaatttgc ggccggtacc caattcgccc tatagtgagt
     3361 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     3421 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     3481 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc
     3541 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3601 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3661 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3721 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     3781 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3841 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3901 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3961 ttttaacaaa atattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg
     4021 tgcggtattt cacaccgcat agatcggcaa gtgcacaaac aatacttaaa taaatactac
     4081 tcagtaataa cctatttctt agcatttttg acgaaatttg ctattttgtt agagtctttt
     4141 acaccatttg tctccacacc tccgcttaca tcaacaccaa taacgccatt taatctaagc
     4201 gcatcaccaa cattttctgg cgtcagtcca ccagctaaca taaaatgtaa gctttcgggg
     4261 ctctcttgcc ttccaaccca gtcagaaatc gagttccaat ccaaaagttc acctgtccca
     4321 cctgcttctg aatcaaacaa gggaataaac gaatgaggtt tctgtgaagc tgcactgagt
     4381 agtatgttgc agtcttttgg aaatacgagt cttttaataa ctggcaaacc gaggaactct
     4441 tggtattctt gccacgactc atctccatgc agttggacga tatcaatgcc gtaatcattg
     4501 accagagcca aaacatcctc cttaggttga ttacgaaaca cgccaaccaa gtatttcgga
     4561 gtgcctgaac tatttttata tgcttttaca agacttgaaa ttttccttgc aataaccggg
     4621 tcaattgttc tctttctatt gggcacacat ataataccca gcaagtcagc atcggaatct
     4681 agagcacatt ctgcggcctc tgtgctctgc aagccgcaaa ctttcaccaa tggaccagaa
     4741 ctacctgtga aattaataac agacatactc caagctgcct ttgtgtgctt aatcacgtat
     4801 actcacgtgc tcaatagtca ccaatgccct ccctcttggc cctctccttt tcttttttcg
     4861 accgaattaa ttcttaatcg gcaaaaaaag aaaagctccg gatcaagatt gtacgtaagg
     4921 tgacaagcta tttttcaata aagaatatct tccactactg ccatctggcg tcataactgc
     4981 aaagtacaca tatattacga tgctgttcta ttaaatgctt cctatattat atatatagta
     5041 atgtcgtgat ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5101 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5161 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5221 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     5281 catgataata atggtttctt agacggatcg cttgcctgta acttacacgc gcctcgtatc
     5341 ttttaatgat ggaataattt gggaatttac tctgtgttta tttattttta tgttttgtat
     5401 ttggatttta gaaagtaaat aaagaaggta gaagagttac ggaatgaaga aaaaaaaata
     5461 aacaaaggtt taaaaaattt caacaaaaag cgtactttac atatatattt attagacaag
     5521 aaaagcagat taaatagata tacattcgat taacgataag taaaatgtaa aatcacagga
     5581 ttttcgtgtg tggtcttcta cacagacaag atgaaacaat tcggcattaa tacctgagag
     5641 caggaagagc aagataaaag gtagtatttg ttggcgatcc ccctagagtc ttttacatct
     5701 tcggaaaaca aaaactattt tttctttaat ttcttttttt actttctatt tttaatttat
     5761 atatttatat taaaaaattt aaattataat tatttttata gcacgtgatg aaaaggaccc
     5821 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5881 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5941 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     6001 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     6061 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     6121 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     6181 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     6241 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     6301 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     6361 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6421 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6481 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6541 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6601 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6661 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6721 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6781 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6841 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6901 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6961 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     7021 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     7081 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     7141 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     7201 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     7261 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     7321 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     7381 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7441 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     7501 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     7561 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     7621 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     7681 tgagtgagct gataccgctc gccgcagccg aacgaccgag c
//
