LOCUS       sequence-335152-        7943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. cerevisiae (yeast) vector for constitutive expression of 
            3xFLAG-RBM28 delta Exon 5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7943)
  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 7943)
  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 dE5
FEATURES             Location/Qualifiers
     source          1..7943
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..38
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    155..176
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(191..196,197..214,215..221)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    229..245
                     /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     234..256
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     253..269
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     253..269
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     288..308
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        290..308
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        333..976
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     870..893
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     982..998
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     982..998
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1015..1080
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    1081..1105
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3296..3320)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      3323..3570
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3339..3357)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3588..3607)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3589..3607)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3617..3634)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3617..3633)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3626..3648)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3774..4229
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3861..3880)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4071..4092
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4332..5006)
                     /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(5007..5287)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(5308..5327)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5427..5449
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5487..5505)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5546..6049
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6086..6190
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6191..6259,6260..7051)
                     /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(6409..6428)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7222..7810
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7711..7730
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
       61 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
      121 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
      181 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      241 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
      301 actaaaggga acaaaagctg gagctcagtt tatcattatc aatactcgcc atttcaaaga
      361 atacgtaaat aattaatagt agtgattttc ctaactttat ttagtcaaaa aattagcctt
      421 ttaattctgc tgtaacccgt acatgcccaa aatagggggc gggttacaca gaatatataa
      481 catcgtaggt gtctgggtga acagtttatt cctggcatcc actaaatata atggagcccg
      541 ctttttaagc tggcatccag aaaaaaaaag aatcccagca ccaaaatatt gttttcttca
      601 ccaaccatca gttcataggt ccattctctt agcgcaacta cagagaacag gggcacaaac
      661 aggcaaaaaa cgggcacaac ctcaatggag tgatgcaacc tgcctggagt aaatgatgac
      721 acaaggcaat tgacccacgc atgtatctat ctcattttct tacaccttct attaccttct
      781 gctctctctg atttggaaaa agctgaaaaa aaaggttgaa accagttccc tgaaattatt
      841 cccctacttg actaataagt atataaagac ggtaggtatt gattgtaatt ctgtaaatct
      901 atttcttaaa cttcttaaat tctactttta tagttagtct tttttttagt tttaaaacac
      961 cagaacttag tttcgacgga ttctagaact agtggatccc ccgggctgca gatggactac
     1021 aaagaccatg acggtgatta taaagatcat gatatcgatt acaaggatga cgatgacaag
     1081 acaagtttgt acaaaaaagc aggctacaaa atggccggcc tgaccttatt tgtgggccgc
     1141 ctcccgccct cggcccgcag tgagcagctg gaggaactgt tcagtcaggt ggggccggtg
     1201 aagcagtgct tcgtggtgac tgaaaaaggg agtaaggcat gtcgaggctt tggctatgtc
     1261 actttttcaa tgctggaaga tgttcagagg gccctcaagg agattaccac ctttgaaggt
     1321 tgcaagatca acgtgactgt tgccaagaaa aaactgagga acaagacaaa ggaaaagggg
     1381 aaaaatgaaa actcagagtg cccaaagaag gagccgaagg ctaaaaaagc caaagtggca
     1441 gataagaaag ccagattaat tattcggaac ctgagcttta agtgttcaga agatgacttg
     1501 aagacagtat ttgctcaatt tggagctgtc ctggaagtaa atatccctag gaaaccaggc
     1561 cggacagtgg ctgtggattg ggccgtggca aaggataaat ataaagatac acagtctgtt
     1621 tctgctatag gtgaggaaaa gagccatgaa tctaaacatc aggaatcagt taaaaagaag
     1681 ggcagagagg aagaggatat ggaagaggaa gaaaacgatg atgatgacga tgatgatgat
     1741 gaagaagatg gggtttttga tgatgaagat gaagaggaag agaatataga atcaaaggtg
     1801 accaagcctg tgcaaattca gaagagagca gtcaagagac cagcccctgc aaaaagcagt
     1861 gatcattctg aggaggacag tgacctagag gaaagcgata gtattgatga tggagaggaa
     1921 ctggctcaga gtgataccag cactgaggag caagaggata aagctgtgca agtctcaaac
     1981 aaaaagaaga ggaaattacc ctctgatgtg aatgaaggga aaactgtttt tatcagaaat
     2041 ctgtcctttg actcagaaga agaagaactt ggggagcttc tccaacagtt tggagaactc
     2101 aaatatgtcc gcattgtctt gcatccagac acagagcatt ctaaaggttg tgcatttgcc
     2161 cagttcatga ctcaagaagc agctcagaaa tgccttctag ctgcttctcc agagaatgag
     2221 gctggtgggc ttaaactgga tggccggcag ctcaaggttg acttggcggt gacccgtgat
     2281 gaggctgcaa agcttcagac gacgaaggtg aagaagccga ctggcacccg gaatctctat
     2341 ctggcccgag aaggcttgat tcgtgctggg acgaaggctg cagagggtgt gagtgctgct
     2401 gatatggcca aaagagaacg gtttgagctg ctgaagcatc agaaactcaa ggaccagaat
     2461 atctttgtct cccgaaccag gctctgcctg cacaatctcc caaaggctgt agatgacaaa
     2521 cagctcagaa agctgctgct gagtgctact agtggagaga aaggggtgcg catcaaggag
     2581 tgtagagtga tgcgagacct caaaggagtt catgggaaca tgaagggtca gtccctgggc
     2641 tacgcctttg cggagttcca agagcacgag catgccctga aagccctccg cctcatcaac
     2701 aacaatccag aaatctttgg gcctctgaag agaccaatag tggagttctc tttagaagat
     2761 cgaagaaaac ttaaaatgaa ggaattaagg atccagcgca gcttgcaaaa aatgagatcc
     2821 aagcctgcaa ctggtgagcc tcagaagggg caaccagagc ctgcaaaaga ccagcaacag
     2881 aaggcagctc aacaccacac agaggaacaa agcaaggtgc ccccagagca gaagagaaag
     2941 gcgggctcta cctcatggac cgggttccag accaaggctg aagtggagca ggtggagctg
     3001 cctgatggaa agaagagaag aaaggtcctg gcgctcccct cacaccgagg ccccaaaatc
     3061 aggttgcggg acaaaggcaa agtgaagccc gtccatccca aaaagccaaa gccacagata
     3121 aaccagtgga agcaggagaa gcagcaatta tcgtccgagc aggtatctag gaaaaaagct
     3181 aagggaaata agacggaaac ccgcttcaac cagctggtcg aacaatataa gcagaaatta
     3241 ttgggacctt ctaaaggagc acctcttgca aagaggagca aatggtttga tagtaaccca
     3301 gctttcttgt acaaagtggt agtcatgtaa ttagttatgt cacgcttaca ttcacgccct
     3361 ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct
     3421 atttattttt ttatagttat gttagtatta agaacgttat ttatatttca aatttttctt
     3481 ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag
     3541 gttttgggac gctcgaaggc tttaatttgc ggccggtacc caattcgccc tatagtgagt
     3601 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     3661 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     3721 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc
     3781 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3841 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3901 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3961 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     4021 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4081 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     4141 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4201 ttttaacaaa atattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg
     4261 tgcggtattt cacaccgcat agatcggcaa gtgcacaaac aatacttaaa taaatactac
     4321 tcagtaataa cctatttctt agcatttttg acgaaatttg ctattttgtt agagtctttt
     4381 acaccatttg tctccacacc tccgcttaca tcaacaccaa taacgccatt taatctaagc
     4441 gcatcaccaa cattttctgg cgtcagtcca ccagctaaca taaaatgtaa gctttcgggg
     4501 ctctcttgcc ttccaaccca gtcagaaatc gagttccaat ccaaaagttc acctgtccca
     4561 cctgcttctg aatcaaacaa gggaataaac gaatgaggtt tctgtgaagc tgcactgagt
     4621 agtatgttgc agtcttttgg aaatacgagt cttttaataa ctggcaaacc gaggaactct
     4681 tggtattctt gccacgactc atctccatgc agttggacga tatcaatgcc gtaatcattg
     4741 accagagcca aaacatcctc cttaggttga ttacgaaaca cgccaaccaa gtatttcgga
     4801 gtgcctgaac tatttttata tgcttttaca agacttgaaa ttttccttgc aataaccggg
     4861 tcaattgttc tctttctatt gggcacacat ataataccca gcaagtcagc atcggaatct
     4921 agagcacatt ctgcggcctc tgtgctctgc aagccgcaaa ctttcaccaa tggaccagaa
     4981 ctacctgtga aattaataac agacatactc caagctgcct ttgtgtgctt aatcacgtat
     5041 actcacgtgc tcaatagtca ccaatgccct ccctcttggc cctctccttt tcttttttcg
     5101 accgaattaa ttcttaatcg gcaaaaaaag aaaagctccg gatcaagatt gtacgtaagg
     5161 tgacaagcta tttttcaata aagaatatct tccactactg ccatctggcg tcataactgc
     5221 aaagtacaca tatattacga tgctgttcta ttaaatgctt cctatattat atatatagta
     5281 atgtcgtgat ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5341 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5401 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5461 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     5521 catgataata atggtttctt agacggatcg cttgcctgta acttacacgc gcctcgtatc
     5581 ttttaatgat ggaataattt gggaatttac tctgtgttta tttattttta tgttttgtat
     5641 ttggatttta gaaagtaaat aaagaaggta gaagagttac ggaatgaaga aaaaaaaata
     5701 aacaaaggtt taaaaaattt caacaaaaag cgtactttac atatatattt attagacaag
     5761 aaaagcagat taaatagata tacattcgat taacgataag taaaatgtaa aatcacagga
     5821 ttttcgtgtg tggtcttcta cacagacaag atgaaacaat tcggcattaa tacctgagag
     5881 caggaagagc aagataaaag gtagtatttg ttggcgatcc ccctagagtc ttttacatct
     5941 tcggaaaaca aaaactattt tttctttaat ttcttttttt actttctatt tttaatttat
     6001 atatttatat taaaaaattt aaattataat tatttttata gcacgtgatg aaaaggaccc
     6061 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     6121 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     6181 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     6241 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     6301 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     6361 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     6421 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     6481 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     6541 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     6601 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6661 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6721 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6781 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6841 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6901 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6961 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     7021 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     7081 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     7141 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     7201 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     7261 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     7321 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     7381 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     7441 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     7501 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     7561 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     7621 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7681 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     7741 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     7801 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     7861 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     7921 tgagtgagct gataccgctc gcc
//
