LOCUS       sequence-335135-        8036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. cerevisiae (yeast) vector for constitutive expression of 
            3xFLAG-RBM28 WT.
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 WT
FEATURES             Location/Qualifiers
     source          1..8036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     54..71
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    188..209
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(224..229,230..247,248..254)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    262..278
                     /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     267..289
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     286..302
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     286..302
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     321..341
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        323..341
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        366..1009
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     903..926
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     1015..1031
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1015..1031
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1048..1113
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    1114..1138
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3422..3446)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      3449..3696
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3465..3483)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3714..3733)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3715..3733)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3743..3760)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3743..3759)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3752..3774)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3900..4355
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3987..4006)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4197..4218
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4458..5132)
                     /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(5133..5413)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(5434..5453)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5553..5575
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5613..5631)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5672..6175
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6212..6316
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6317..6385,6386..7177)
                     /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(6535..6554)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7348..7936
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7837..7856
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
       61 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
      121 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      181 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
      241 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
      301 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctca
      361 gtttatcatt atcaatactc gccatttcaa agaatacgta aataattaat agtagtgatt
      421 ttcctaactt tatttagtca aaaaattagc cttttaattc tgctgtaacc cgtacatgcc
      481 caaaataggg ggcgggttac acagaatata taacatcgta ggtgtctggg tgaacagttt
      541 attcctggca tccactaaat ataatggagc ccgcttttta agctggcatc cagaaaaaaa
      601 aagaatccca gcaccaaaat attgttttct tcaccaacca tcagttcata ggtccattct
      661 cttagcgcaa ctacagagaa caggggcaca aacaggcaaa aaacgggcac aacctcaatg
      721 gagtgatgca acctgcctgg agtaaatgat gacacaaggc aattgaccca cgcatgtatc
      781 tatctcattt tcttacacct tctattacct tctgctctct ctgatttgga aaaagctgaa
      841 aaaaaaggtt gaaaccagtt ccctgaaatt attcccctac ttgactaata agtatataaa
      901 gacggtaggt attgattgta attctgtaaa tctatttctt aaacttctta aattctactt
      961 ttatagttag tctttttttt agttttaaaa caccagaact tagtttcgac ggattctaga
     1021 actagtggat cccccgggct gcagatggac tacaaagacc atgacggtga ttataaagat
     1081 catgatatcg attacaagga tgacgatgac aagacaagtt tgtacaaaaa agcaggctac
     1141 aaaatggccg gcctgacctt atttgtgggc cgcctcccgc cctcggcccg cagtgagcag
     1201 ctggaggaac tgttcagtca ggtggggccg gtgaagcagt gcttcgtggt gactgaaaaa
     1261 gggagtaagg catgtcgagg ctttggctat gtcacttttt caatgctgga agatgttcag
     1321 agggccctca aggagattac cacctttgaa ggttgcaaga tcaacgtgac tgttgccaag
     1381 aaaaaactga ggaacaagac aaaggaaaag gggaaaaatg aaaactcaga gtgcccaaag
     1441 aaggagccga aggctaaaaa agccaaagtg gcagataaga aagccagatt aattattcgg
     1501 aacctgagct ttaagtgttc agaagatgac ttgaagacag tatttgctca atttggagct
     1561 gtcctggaag taaatatccc taggaaacca gatgggaaga tgcgcggttt tggttttgtt
     1621 cagttcaaaa acctcctaga agcaggtaaa gctctcaaag gcatgaacat gaaagagata
     1681 aaaggccgga cagtggctgt ggattgggcc gtggcaaagg ataaatataa agatacacag
     1741 tctgtttctg ctataggtga ggaaaagagc catgaatcta aacatcagga atcagttaaa
     1801 aagaagggca gagaggaaga ggatatggaa gaggaagaaa acgatgatga tgacgatgat
     1861 gatgatgaag aagatggggt ttttgatgat gaagatgaag aggaagagaa tatagaatca
     1921 aaggtgacca agcctgtgca aattcagaag agagcagtca agagaccagc ccctgcaaaa
     1981 agcagtgatc attctgagga ggacagtgac ctagaggaaa gcgatagtat tgatgatgga
     2041 gaggaactgg ctcagagtga taccagcact gaggagcaag aggataaagc tgtgcaagtc
     2101 tcaaacaaaa agaagaggaa attaccctct gatgtgaatg aagggaaaac tgtttttatc
     2161 agaaatctgt cctttgactc agaagaagaa gaacttgggg agcttctcca acagtttgga
     2221 gaactcaaat atgtccgcat tgtcttgcat ccagacacag agcattctaa aggttgtgca
     2281 tttgcccagt tcatgactca agaagcagct cagaaatgcc ttctagctgc ttctccagag
     2341 aatgaggctg gtgggcttaa actggatggc cggcagctca aggttgactt ggcggtgacc
     2401 cgtgatgagg ctgcaaagct tcagacgacg aaggtgaaga agccgactgg cacccggaat
     2461 ctctatctgg cccgagaagg cttgattcgt gctgggacga aggctgcaga gggtgtgagt
     2521 gctgctgata tggccaaaag agaacggttt gagctgctga agcatcagaa actcaaggac
     2581 cagaatatct ttgtctcccg aaccaggctc tgcctgcaca atctcccaaa ggctgtagat
     2641 gacaaacagc tcagaaagct gctgctgagt gctactagtg gagagaaagg ggtgcgcatc
     2701 aaggagtgta gagtgatgcg agacctcaaa ggagttcatg ggaacatgaa gggtcagtcc
     2761 ctgggctacg cctttgcgga gttccaagag cacgagcatg ccctgaaagc cctccgcctc
     2821 atcaacaaca atccagaaat ctttgggcct ctgaagagac caatagtgga gttctcttta
     2881 gaagatcgaa gaaaacttaa aatgaaggaa ttaaggatcc agcgcagctt gcaaaaaatg
     2941 agatccaagc ctgcaactgg tgagcctcag aaggggcaac cagagcctgc aaaagaccag
     3001 caacagaagg cagctcaaca ccacacagag gaacaaagca aggtgccccc agagcagaag
     3061 agaaaggcgg gctctacctc atggaccggg ttccagacca aggctgaagt ggagcaggtg
     3121 gagctgcctg atggaaagaa gagaagaaag gtcctggcgc tcccctcaca ccgaggcccc
     3181 aaaatcaggt tgcgggacaa aggcaaagtg aagcccgtcc atcccaaaaa gccaaagcca
     3241 cagataaacc agtggaagca ggagaagcag caattatcgt ccgagcaggt atctaggaaa
     3301 aaagctaagg gaaataagac ggaaacccgc ttcaaccagc tggtcgaaca atataagcag
     3361 aaattattgg gaccttctaa aggagcacct cttgcaaaga ggagcaaatg gtttgatagt
     3421 aacccagctt tcttgtacaa agtggtagtc atgtaattag ttatgtcacg cttacattca
     3481 cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag
     3541 gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt
     3601 tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt
     3661 gagaaggttt tgggacgctc gaaggcttta atttgcggcc ggtacccaat tcgccctata
     3721 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     3781 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     3841 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     3901 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3961 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4021 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4081 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     4141 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4201 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     4261 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4321 cgcgaatttt aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg
     4381 catctgtgcg gtatttcaca ccgcatagat cggcaagtgc acaaacaata cttaaataaa
     4441 tactactcag taataaccta tttcttagca tttttgacga aatttgctat tttgttagag
     4501 tcttttacac catttgtctc cacacctccg cttacatcaa caccaataac gccatttaat
     4561 ctaagcgcat caccaacatt ttctggcgtc agtccaccag ctaacataaa atgtaagctt
     4621 tcggggctct cttgccttcc aacccagtca gaaatcgagt tccaatccaa aagttcacct
     4681 gtcccacctg cttctgaatc aaacaaggga ataaacgaat gaggtttctg tgaagctgca
     4741 ctgagtagta tgttgcagtc ttttggaaat acgagtcttt taataactgg caaaccgagg
     4801 aactcttggt attcttgcca cgactcatct ccatgcagtt ggacgatatc aatgccgtaa
     4861 tcattgacca gagccaaaac atcctcctta ggttgattac gaaacacgcc aaccaagtat
     4921 ttcggagtgc ctgaactatt tttatatgct tttacaagac ttgaaatttt ccttgcaata
     4981 accgggtcaa ttgttctctt tctattgggc acacatataa tacccagcaa gtcagcatcg
     5041 gaatctagag cacattctgc ggcctctgtg ctctgcaagc cgcaaacttt caccaatgga
     5101 ccagaactac ctgtgaaatt aataacagac atactccaag ctgcctttgt gtgcttaatc
     5161 acgtatactc acgtgctcaa tagtcaccaa tgccctccct cttggccctc tccttttctt
     5221 ttttcgaccg aattaattct taatcggcaa aaaaagaaaa gctccggatc aagattgtac
     5281 gtaaggtgac aagctatttt tcaataaaga atatcttcca ctactgccat ctggcgtcat
     5341 aactgcaaag tacacatata ttacgatgct gttctattaa atgcttccta tattatatat
     5401 atagtaatgt cgtgatctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5461 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5521 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5581 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5641 taatgtcatg ataataatgg tttcttagac ggatcgcttg cctgtaactt acacgcgcct
     5701 cgtatctttt aatgatggaa taatttggga atttactctg tgtttattta tttttatgtt
     5761 ttgtatttgg attttagaaa gtaaataaag aaggtagaag agttacggaa tgaagaaaaa
     5821 aaaataaaca aaggtttaaa aaatttcaac aaaaagcgta ctttacatat atatttatta
     5881 gacaagaaaa gcagattaaa tagatataca ttcgattaac gataagtaaa atgtaaaatc
     5941 acaggatttt cgtgtgtggt cttctacaca gacaagatga aacaattcgg cattaatacc
     6001 tgagagcagg aagagcaaga taaaaggtag tatttgttgg cgatccccct agagtctttt
     6061 acatcttcgg aaaacaaaaa ctattttttc tttaatttct ttttttactt tctattttta
     6121 atttatatat ttatattaaa aaatttaaat tataattatt tttatagcac gtgatgaaaa
     6181 ggacccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     6241 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     6301 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     6361 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     6421 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     6481 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     6541 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     6601 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     6661 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6721 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6781 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6841 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6901 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     6961 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     7021 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     7081 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     7141 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     7201 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     7261 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     7321 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     7381 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     7441 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     7501 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7561 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7621 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7681 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7741 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7801 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7861 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7921 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     7981 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgta
//
