LOCUS       sequence-335138-        8036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. cerevisiae (yeast) vector for constitutive expression of 
            3xFLAG-RBM28 L351P.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8036)
  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 8036)
  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-RBM28 L351P
FEATURES             Location/Qualifiers
     source          1..8036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..186
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(187..255,256..1047)
                     /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(405..424)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1218..1806
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1707..1726
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1960..1977
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2094..2115
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2130..2135,2136..2153,2154..2160)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2168..2184
                     /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     2173..2195
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2192..2208
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2192..2208
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2227..2247
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2229..2247
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2272..2915
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     2809..2832
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     2921..2937
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2921..2937
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             2954..3019
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    3020..3044
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5328..5352)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5355..5602
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5371..5389)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5620..5639)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5621..5639)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5649..5666)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5649..5665)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5658..5680)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5806..6261
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5893..5912)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6103..6124
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6364..7038)
                     /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(7039..7319)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(7340..7359)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7459..7481
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7519..7537)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    join(7578..8036,1..45)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 ttatattaaa aaatttaaat tataattatt tttatagcac gtgatgaaaa ggacccaggt
       61 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
      121 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
      181 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
      241 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
      301 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
      361 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
      421 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
      481 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
      541 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
      601 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
      661 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
      721 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
      781 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
      841 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
      901 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
      961 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     1021 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     1081 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     1141 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     1201 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     1261 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1321 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     1381 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     1441 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     1501 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     1561 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     1621 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     1681 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     1741 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     1801 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     1861 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     1921 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     1981 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     2041 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     2101 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     2161 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     2221 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctcagttt atcattatca
     2281 atactcgcca tttcaaagaa tacgtaaata attaatagta gtgattttcc taactttatt
     2341 tagtcaaaaa attagccttt taattctgct gtaacccgta catgcccaaa atagggggcg
     2401 ggttacacag aatatataac atcgtaggtg tctgggtgaa cagtttattc ctggcatcca
     2461 ctaaatataa tggagcccgc tttttaagct ggcatccaga aaaaaaaaga atcccagcac
     2521 caaaatattg ttttcttcac caaccatcag ttcataggtc cattctctta gcgcaactac
     2581 agagaacagg ggcacaaaca ggcaaaaaac gggcacaacc tcaatggagt gatgcaacct
     2641 gcctggagta aatgatgaca caaggcaatt gacccacgca tgtatctatc tcattttctt
     2701 acaccttcta ttaccttctg ctctctctga tttggaaaaa gctgaaaaaa aaggttgaaa
     2761 ccagttccct gaaattattc ccctacttga ctaataagta tataaagacg gtaggtattg
     2821 attgtaattc tgtaaatcta tttcttaaac ttcttaaatt ctacttttat agttagtctt
     2881 ttttttagtt ttaaaacacc agaacttagt ttcgacggat tctagaacta gtggatcccc
     2941 cgggctgcag atggactaca aagaccatga cggtgattat aaagatcatg atatcgatta
     3001 caaggatgac gatgacaaga caagtttgta caaaaaagca ggctacaaaa tggccggcct
     3061 gaccttattt gtgggccgcc tcccgccctc ggcccgcagt gagcagctgg aggaactgtt
     3121 cagtcaggtg gggccggtga agcagtgctt cgtggtgact gaaaaaggga gtaaggcatg
     3181 tcgaggcttt ggctatgtca ctttttcaat gctggaagat gttcagaggg ccctcaagga
     3241 gattaccacc tttgaaggtt gcaagatcaa cgtgactgtt gccaagaaaa aactgaggaa
     3301 caagacaaag gaaaagggga aaaatgaaaa ctcagagtgc ccaaagaagg agccgaaggc
     3361 taaaaaagcc aaagtggcag ataagaaagc cagattaatt attcggaacc tgagctttaa
     3421 gtgttcagaa gatgacttga agacagtatt tgctcaattt ggagctgtcc tggaagtaaa
     3481 tatccctagg aaaccagatg ggaagatgcg cggttttggt tttgttcagt tcaaaaacct
     3541 cctagaagca ggtaaagctc tcaaaggcat gaacatgaaa gagataaaag gccggacagt
     3601 ggctgtggat tgggccgtgg caaaggataa atataaagat acacagtctg tttctgctat
     3661 aggtgaggaa aagagccatg aatctaaaca tcaggaatca gttaaaaaga agggcagaga
     3721 ggaagaggat atggaagagg aagaaaacga tgatgatgac gatgatgatg atgaagaaga
     3781 tggggttttt gatgatgaag atgaagagga agagaatata gaatcaaagg tgaccaagcc
     3841 tgtgcaaatt cagaagagag cagtcaagag accagcccct gcaaaaagca gtgatcattc
     3901 tgaggaggac agtgacctag aggaaagcga tagtattgat gatggagagg aactggctca
     3961 gagtgatacc agcactgagg agcaagagga taaagctgtg caagtctcaa acaaaaagaa
     4021 gaggaaatta ccctctgatg tgaatgaagg gaaaactgtt tttatcagaa atctgtcctt
     4081 tgactcagaa gaagaagaac ctggggagct tctccaacag tttggagaac tcaaatatgt
     4141 ccgcattgtc ttgcatccag acacagagca ttctaaaggt tgtgcatttg cccagttcat
     4201 gactcaagaa gcagctcaga aatgccttct agctgcttct ccagagaatg aggctggtgg
     4261 gcttaaactg gatggccggc agctcaaggt tgacttggcg gtgacccgtg atgaggctgc
     4321 aaagcttcag acgacgaagg tgaagaagcc gactggcacc cggaatctct atctggcccg
     4381 agaaggcttg attcgtgctg ggacgaaggc tgcagagggt gtgagtgctg ctgatatggc
     4441 caaaagagaa cggtttgagc tgctgaagca tcagaaactc aaggaccaga atatctttgt
     4501 ctcccgaacc aggctctgcc tgcacaatct cccaaaggct gtagatgaca aacagctcag
     4561 aaagctgctg ctgagtgcta ctagtggaga gaaaggggtg cgcatcaagg agtgtagagt
     4621 gatgcgagac ctcaaaggag ttcatgggaa catgaagggt cagtccctgg gctacgcctt
     4681 tgcggagttc caagagcacg agcatgccct gaaagccctc cgcctcatca acaacaatcc
     4741 agaaatcttt gggcctctga agagaccaat agtggagttc tctttagaag atcgaagaaa
     4801 acttaaaatg aaggaattaa ggatccagcg cagcttgcaa aaaatgagat ccaagcctgc
     4861 aactggtgag cctcagaagg ggcaaccaga gcctgcaaaa gaccagcaac agaaggcagc
     4921 tcaacaccac acagaggaac aaagcaaggt gcccccagag cagaagagaa aggcgggctc
     4981 tacctcatgg accgggttcc agaccaaggc tgaagtggag caggtggagc tgcctgatgg
     5041 aaagaagaga agaaaggtcc tggcgctccc ctcacaccga ggccccaaaa tcaggttgcg
     5101 ggacaaaggc aaagtgaagc ccgtccatcc caaaaagcca aagccacaga taaaccagtg
     5161 gaagcaggag aagcagcaat tatcgtccga gcaggtatct aggaaaaaag ctaagggaaa
     5221 taagacggaa acccgcttca accagctggt cgaacaatat aagcagaaat tattgggacc
     5281 ttctaaagga gcacctcttg caaagaggag caaatggttt gatagtaacc cagctttctt
     5341 gtacaaagtg gtagtcatgt aattagttat gtcacgctta cattcacgcc ctccccccac
     5401 atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt
     5461 ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct
     5521 gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg
     5581 acgctcgaag gctttaattt gcggccggta cccaattcgc cctatagtga gtcgtattac
     5641 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     5701 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     5761 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg
     5821 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     5881 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     5941 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     6001 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     6061 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     6121 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     6181 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     6241 aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     6301 ttcacaccgc atagatcggc aagtgcacaa acaatactta aataaatact actcagtaat
     6361 aacctatttc ttagcatttt tgacgaaatt tgctattttg ttagagtctt ttacaccatt
     6421 tgtctccaca cctccgctta catcaacacc aataacgcca tttaatctaa gcgcatcacc
     6481 aacattttct ggcgtcagtc caccagctaa cataaaatgt aagctttcgg ggctctcttg
     6541 ccttccaacc cagtcagaaa tcgagttcca atccaaaagt tcacctgtcc cacctgcttc
     6601 tgaatcaaac aagggaataa acgaatgagg tttctgtgaa gctgcactga gtagtatgtt
     6661 gcagtctttt ggaaatacga gtcttttaat aactggcaaa ccgaggaact cttggtattc
     6721 ttgccacgac tcatctccat gcagttggac gatatcaatg ccgtaatcat tgaccagagc
     6781 caaaacatcc tccttaggtt gattacgaaa cacgccaacc aagtatttcg gagtgcctga
     6841 actattttta tatgctttta caagacttga aattttcctt gcaataaccg ggtcaattgt
     6901 tctctttcta ttgggcacac atataatacc cagcaagtca gcatcggaat ctagagcaca
     6961 ttctgcggcc tctgtgctct gcaagccgca aactttcacc aatggaccag aactacctgt
     7021 gaaattaata acagacatac tccaagctgc ctttgtgtgc ttaatcacgt atactcacgt
     7081 gctcaatagt caccaatgcc ctccctcttg gccctctcct tttctttttt cgaccgaatt
     7141 aattcttaat cggcaaaaaa agaaaagctc cggatcaaga ttgtacgtaa ggtgacaagc
     7201 tatttttcaa taaagaatat cttccactac tgccatctgg cgtcataact gcaaagtaca
     7261 catatattac gatgctgttc tattaaatgc ttcctatatt atatatatag taatgtcgtg
     7321 atctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     7381 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     7441 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     7501 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     7561 taatggtttc ttagacggat cgcttgcctg taacttacac gcgcctcgta tcttttaatg
     7621 atggaataat ttgggaattt actctgtgtt tatttatttt tatgttttgt atttggattt
     7681 tagaaagtaa ataaagaagg tagaagagtt acggaatgaa gaaaaaaaaa taaacaaagg
     7741 tttaaaaaat ttcaacaaaa agcgtacttt acatatatat ttattagaca agaaaagcag
     7801 attaaataga tatacattcg attaacgata agtaaaatgt aaaatcacag gattttcgtg
     7861 tgtggtcttc tacacagaca agatgaaaca attcggcatt aatacctgag agcaggaaga
     7921 gcaagataaa aggtagtatt tgttggcgat ccccctagag tcttttacat cttcggaaaa
     7981 caaaaactat tttttcttta atttcttttt ttactttcta tttttaattt atatat
//
