LOCUS       sequence-328262-        6752 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian vector for constitutive expression of RBM28 Exon 5 GT>A 
            minigene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6752)
  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 6752)
  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: pcDNA5/FRT/TO - RBM28 E5 GT>A minigene
FEATURES             Location/Qualifiers
     source          1..6752
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(2704..2721)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2710..2934
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3067..3086)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3218..3265
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3273..4295
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3273..4295
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4425..4546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4462..4481)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4516..4535
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4595..4611)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4595..4611)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4608..4630)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4619..4635
                     /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)."
     promoter        complement(join(4643..4649,4650..4667,4668..4673))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4688..4709
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4826..4843)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4997..5585)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5077..5096)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5756..6547,6548..6616))
                     /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     6379..6398
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6617..6721)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtacct gttcagaaga tgacttgaag acagtatttg
     1021 ctcaatttgg agctgtcctg gaagtaaata tccctaggaa accaggtact aataaacctt
     1081 cacttggtgt ggattacgtt agttaacata ttacccttga acaaacatat atgatatata
     1141 tgaagtattt ggcacagtgc ttcacatcta atgaaaaccc aaataatggt tttattcccc
     1201 tgatgatatt ttctgttgga tcagaattag ttttagaagg cgagttagtt tgagaaagtg
     1261 ttttgctctc cccaagtcca agtcagagta tactcagtaa gaagatgtaa gcaattgctt
     1321 tggggaaggg cctgacctgc tggcagaatt tgggccatct tgtgggcctg aattgcagta
     1381 atttacttct tagtattctg gctacctcct gctgcctctc acttctttct ttgtgcattt
     1441 gcacacagat gggaagatgc gcggttttgg ttttgttcag ttcaaaaacc tcctagaagc
     1501 aggtaaagct ctcaaaggca tgaacatgaa agagataaaa gaaagctttc tatacccata
     1561 tcattgaccc agatatttct ggtgttttgg tgaattagtg tagtaaccca ctttctgcat
     1621 tttcaaaaga tgtttgcatt attaagagtc tcattgctaa tacggtagct atttaggtga
     1681 aatactctag aacttgatct tggggttaaa cttgaaagaa cccttgccag tattctttct
     1741 tttatctggt gagagtgtcc ttttaaatcc ctagtgaaaa atgtccctga gataattgct
     1801 gatagatgca ggttggtatc tagattcctc cttggttata tcactgttca gcatctgtgg
     1861 gctatttatt aaactgactg tgatttatat attgacttat caagtactgt gcctaaagaa
     1921 gttattgccg gccaggtgcg gtggctcaca cctgtaatcc tagcactttg ggaggccgag
     1981 gaggcaggtg gatcacctga ggtcaggagt tcaagaccag cctggtcaac atggcgaaac
     2041 cccgtctcta ctgaaaatac aaaaattagc caggtgtggt ggtgcatgct tgtaatccca
     2101 gctactcaag aggctgaggc aggagaatcg cttgaacctg gaggcggagg ttgcagtgaa
     2161 ctgagatggc accactgcac tccagcctgg gcgacagagc gagactgtgt ctcaaaaaaa
     2221 aaaaaaagaa gttattgccg tagagatgtc ctagcttcca ggtagggtgc agatgattcc
     2281 agaatgcctg ttttgattcc tgatagtcta actggaaaca tagttctaat tctttatttt
     2341 tttactccat cttatcccag gtgtggtggc tatgtagaaa ggcaaaattt ctgttcattg
     2401 ataaaaaggt taagggttaa aaggagtagg cagtgactag gagctttatt ttaagaccag
     2461 cattccctga attggcagtt tctttgtctc cactggttcc attattgtct gtgatgacta
     2521 ggggtcattt acagaggtag ggtttttaaa aggagagcct ttctgatgcc acttttctgc
     2581 ttgacaggcc ggacagtggc tgtggattgg gccgtggcaa aggataaata taaagataca
     2641 cagtctgttt ctgctatagg cggccgctcg agtctagagg gcccgtttaa acccgctgat
     2701 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     2761 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2821 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     2881 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     2941 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     3001 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3061 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3121 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3181 ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct
     3241 attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg
     3301 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg
     3361 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     3421 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     3481 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca
     3541 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     3601 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     3661 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     3721 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     3781 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     3841 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     3901 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     3961 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     4021 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     4081 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     4141 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     4201 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     4261 gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca
     4321 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4381 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4441 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4501 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4561 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4621 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4681 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4741 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4801 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4861 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4921 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4981 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5041 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5101 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5161 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5221 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5281 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5341 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5401 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5461 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5521 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5581 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5641 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5701 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5761 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5821 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5881 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5941 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6001 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6061 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6121 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6181 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6241 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6301 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6361 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6421 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6481 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6541 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6601 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6661 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6721 gcacatttcc ccgaaaagtg ccacctgacg tc
//
