LOCUS       sequence-335155-        7899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. cerevisiae (yeast) vector for constitutive expression of 
            3xFLAG-RBM28 delta Exon 8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7899)
  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 7899)
  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 dE8
FEATURES             Location/Qualifiers
     source          1..7899
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    69..572
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        609..713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(714..782,783..1574)
                     /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(932..951)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1745..2333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2234..2253
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2487..2504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2621..2642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2657..2662,2663..2680,2681..2687)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2695..2711
                     /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     2700..2722
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2719..2735
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2719..2735
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2754..2774
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2756..2774
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2799..3442
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3336..3359
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     3448..3464
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3448..3464
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             3481..3546
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    3547..3571
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5718..5742)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      5745..5992
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5761..5779)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(6010..6029)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6011..6029)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6039..6056)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6039..6055)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6048..6070)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6196..6651
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6283..6302)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6493..6514
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6754..7428)
                     /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(7429..7709)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(7730..7749)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7849..7871
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
      121 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
      181 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
      241 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
      301 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
      361 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
      421 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
      481 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
      541 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
      601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1141 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1861 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     1921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2521 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2581 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     2641 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     2701 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
     2761 accctcacta aagggaacaa aagctggagc tcagtttatc attatcaata ctcgccattt
     2821 caaagaatac gtaaataatt aatagtagtg attttcctaa ctttatttag tcaaaaaatt
     2881 agccttttaa ttctgctgta acccgtacat gcccaaaata gggggcgggt tacacagaat
     2941 atataacatc gtaggtgtct gggtgaacag tttattcctg gcatccacta aatataatgg
     3001 agcccgcttt ttaagctggc atccagaaaa aaaaagaatc ccagcaccaa aatattgttt
     3061 tcttcaccaa ccatcagttc ataggtccat tctcttagcg caactacaga gaacaggggc
     3121 acaaacaggc aaaaaacggg cacaacctca atggagtgat gcaacctgcc tggagtaaat
     3181 gatgacacaa ggcaattgac ccacgcatgt atctatctca ttttcttaca ccttctatta
     3241 ccttctgctc tctctgattt ggaaaaagct gaaaaaaaag gttgaaacca gttccctgaa
     3301 attattcccc tacttgacta ataagtatat aaagacggta ggtattgatt gtaattctgt
     3361 aaatctattt cttaaacttc ttaaattcta cttttatagt tagtcttttt tttagtttta
     3421 aaacaccaga acttagtttc gacggattct agaactagtg gatcccccgg gctgcagatg
     3481 gactacaaag accatgacgg tgattataaa gatcatgata tcgattacaa ggatgacgat
     3541 gacaagacaa gtttgtacaa aaaagcaggc tacaaaatgg ccggcctgac cttatttgtg
     3601 ggccgcctcc cgccctcggc ccgcagtgag cagctggagg aactgttcag tcaggtgggg
     3661 ccggtgaagc agtgcttcgt ggtgactgaa aaagggagta aggcatgtcg aggctttggc
     3721 tatgtcactt tttcaatgct ggaagatgtt cagagggccc tcaaggagat taccaccttt
     3781 gaaggttgca agatcaacgt gactgttgcc aagaaaaaac tgaggaacaa gacaaaggaa
     3841 aaggggaaaa atgaaaactc agagtgccca aagaaggagc cgaaggctaa aaaagccaaa
     3901 gtggcagata agaaagccag attaattatt cggaacctga gctttaagtg ttcagaagat
     3961 gacttgaaga cagtatttgc tcaatttgga gctgtcctgg aagtaaatat ccctaggaaa
     4021 ccagatggga agatgcgcgg ttttggtttt gttcagttca aaaacctcct agaagcaggt
     4081 aaagctctca aaggcatgaa catgaaagag ataaaaggcc ggacagtggc tgtggattgg
     4141 gccgtggcaa aggataaata taaagataca cagtctgttt ctgctatagg tgaggaaaag
     4201 agccatgaat ctaaacatca ggaatcagtt aaaaagaagg gcagagagga agaggatatg
     4261 gaagaggaag aaaacgatga tgatgacgat gatgatgatg aagaagatgg ggtttttgat
     4321 gatgaagatg aagaggaaga gaatatagaa tcaaaggtga ccaagcctgt gcaaattcag
     4381 aagagctgtg caagtctcaa acaaaaagaa gaggaaatta ccctctgatg tgaatgaagg
     4441 gaaaactgtt tttatcagaa atctgtcctt tgactcagaa gaagaagaac ttggggagct
     4501 tctccaacag tttggagaac tcaaatatgt ccgcattgtc ttgcatccag acacagagca
     4561 ttctaaaggt tgtgcatttg cccagttcat gactcaagaa gcagctcaga aatgccttct
     4621 agctgcttct ccagagaatg aggctggtgg gcttaaactg gatggccggc agctcaaggt
     4681 tgacttggcg gtgacccgtg atgaggctgc aaagcttcag acgacgaagg tgaagaagcc
     4741 gactggcacc cggaatctct atctggcccg agaaggcttg attcgtgctg ggacgaaggc
     4801 tgcagagggt gtgagtgctg ctgatatggc caaaagagaa cggtttgagc tgctgaagca
     4861 tcagaaactc aaggaccaga atatctttgt ctcccgaacc aggctctgcc tgcacaatct
     4921 cccaaaggct gtagatgaca aacagctcag aaagctgctg ctgagtgcta ctagtggaga
     4981 gaaaggggtg cgcatcaagg agtgtagagt gatgcgagac ctcaaaggag ttcatgggaa
     5041 catgaagggt cagtccctgg gctacgcctt tgcggagttc caagagcacg agcatgccct
     5101 gaaagccctc cgcctcatca acaacaatcc agaaatcttt gggcctctga agagaccaat
     5161 agtggagttc tctttagaag atcgaagaaa acttaaaatg aaggaattaa ggatccagcg
     5221 cagcttgcaa aaaatgagat ccaagcctgc aactggtgag cctcagaagg ggcaaccaga
     5281 gcctgcaaaa gaccagcaac agaaggcagc tcaacaccac acagaggaac aaagcaaggt
     5341 gcccccagag cagaagagaa aggcgggctc tacctcatgg accgggttcc agaccaaggc
     5401 tgaagtggag caggtggagc tgcctgatgg aaagaagaga agaaaggtcc tggcgctccc
     5461 ctcacaccga ggccccaaaa tcaggttgcg ggacaaaggc aaagtgaagc ccgtccatcc
     5521 caaaaagcca aagccacaga taaaccagtg gaagcaggag aagcagcaat tatcgtccga
     5581 gcaggtatct aggaaaaaag ctaagggaaa taagacggaa acccgcttca accagctggt
     5641 cgaacaatat aagcagaaat tattgggacc ttctaaagga gcacctcttg caaagaggag
     5701 caaatggttt gatagtaacc cagctttctt gtacaaagtg gtagtcatgt aattagttat
     5761 gtcacgctta cattcacgcc ctccccccac atccgctcta accgaaaagg aaggagttag
     5821 acaacctgaa gtctaggtcc ctatttattt ttttatagtt atgttagtat taagaacgtt
     5881 atttatattt caaatttttc ttttttttct gtacagacgc gtgtacgcat gtaacattat
     5941 actgaaaacc ttgcttgaga aggttttggg acgctcgaag gctttaattt gcggccggta
     6001 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     6061 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     6121 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     6181 ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     6241 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     6301 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     6361 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     6421 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     6481 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     6541 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     6601 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt tcctgatgcg
     6661 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagatcggc aagtgcacaa
     6721 acaatactta aataaatact actcagtaat aacctatttc ttagcatttt tgacgaaatt
     6781 tgctattttg ttagagtctt ttacaccatt tgtctccaca cctccgctta catcaacacc
     6841 aataacgcca tttaatctaa gcgcatcacc aacattttct ggcgtcagtc caccagctaa
     6901 cataaaatgt aagctttcgg ggctctcttg ccttccaacc cagtcagaaa tcgagttcca
     6961 atccaaaagt tcacctgtcc cacctgcttc tgaatcaaac aagggaataa acgaatgagg
     7021 tttctgtgaa gctgcactga gtagtatgtt gcagtctttt ggaaatacga gtcttttaat
     7081 aactggcaaa ccgaggaact cttggtattc ttgccacgac tcatctccat gcagttggac
     7141 gatatcaatg ccgtaatcat tgaccagagc caaaacatcc tccttaggtt gattacgaaa
     7201 cacgccaacc aagtatttcg gagtgcctga actattttta tatgctttta caagacttga
     7261 aattttcctt gcaataaccg ggtcaattgt tctctttcta ttgggcacac atataatacc
     7321 cagcaagtca gcatcggaat ctagagcaca ttctgcggcc tctgtgctct gcaagccgca
     7381 aactttcacc aatggaccag aactacctgt gaaattaata acagacatac tccaagctgc
     7441 ctttgtgtgc ttaatcacgt atactcacgt gctcaatagt caccaatgcc ctccctcttg
     7501 gccctctcct tttctttttt cgaccgaatt aattcttaat cggcaaaaaa agaaaagctc
     7561 cggatcaaga ttgtacgtaa ggtgacaagc tatttttcaa taaagaatat cttccactac
     7621 tgccatctgg cgtcataact gcaaagtaca catatattac gatgctgttc tattaaatgc
     7681 ttcctatatt atatatatag taatgtcgtg atctatggtg cactctcagt acaatctgct
     7741 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     7801 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     7861 tgtgtcagag gttttcaccg tcatcaccga aacgcgcga
//
