LOCUS       sequence-335162-        6755 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 6755)
  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 6755)
  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..6755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        156..535
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        536..739
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     690..710
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    741..759
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    762..780
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     790..814
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(2628..2645)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2634..2858
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2991..3010)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3142..3189
                     /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             3197..4219
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3197..4219
                     /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    4349..4470
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4386..4405)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4440..4459
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4519..4535)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4519..4535)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4532..4554)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4543..4559
                     /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(4567..4573,4574..4591,4592..4597))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4612..4633
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4750..4767)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4921..5509)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5001..5020)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5680..6471,6472..6540))
                     /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     6303..6322
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6541..6645)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6720..6739)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
       61 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
      121 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
      181 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      241 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      301 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      361 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      421 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      481 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      541 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
      601 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      661 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
      721 ggtctatata agcagagctc tccctatcag tgatagagat ctccctatca gtgatagaga
      781 tcgtcgacga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt
      841 tgacctccat agaagacacc gggaccgatc cagcctccgg actctagcgt ttaaacttaa
      901 gcttggtacc tgttcagaag atgacttgaa gacagtattt gctcaatttg gagctgtcct
      961 ggaagtaaat atccctagga aaccaggtac taataaacct tcacttggtg tggattacgt
     1021 tagttaacat attacccttg aacaaacata tatgatatat atgaagtatt tggcacagtg
     1081 cttcacatct aatgaaaacc caaataatgg ttttattccc ctgatgatat tttctgttgg
     1141 atcagaatta gttttagaag gcgagttagt ttgagaaagt gttttgctct ccccaagtcc
     1201 aagtcagagt atactcagta agaagatgta agcaattgct ttggggaagg gcctgacctg
     1261 ctggcagaat ttgggccatc ttgtgggcct gaattgcagt aatttacttc ttagtattct
     1321 ggctacctcc tgctgcctct cacttctttc tttgtgcatt tgcacacaga tgggaagatg
     1381 cgcggttttg gttttgttca gttcaaaaac ctcctagaag caggtaaagc tctcaaaggc
     1441 atgaacatga aagagataaa agaaagcttt ctatacccat atcattgacc cagatatttc
     1501 tggtgttttg gtgaattagt gtagtaaccc actttctgca ttttcaaaag atgtttgcat
     1561 tattaagagt ctcattgcta atacggtagc tatttaggtg aaatactcta gaacttgatc
     1621 ttggggttaa acttgaaaga acccttgcca gtattctttc ttttatctgg tgagagtgtc
     1681 cttttaaatc cctagtgaaa aatgtccctg agataattgc tgatagatgc aggttggtat
     1741 ctagattcct ccttggttat atcactgttc agcatctgtg ggctatttat taaactgact
     1801 gtgatttata tattgactta tcaagtactg tgcctaaaga agttattgcc ggccaggtgc
     1861 ggtggctcac acctgtaatc ctagcacttt gggaggccga ggaggcaggt ggatcacctg
     1921 aggtcaggag ttcaagacca gcctggtcaa catggcgaaa ccccgtctct actgaaaata
     1981 caaaaattag ccaggtgtgg tggtgcatgc ttgtaatccc agctactcaa gaggctgagg
     2041 caggagaatc gcttgaacct ggaggcggag gttgcagtga actgagatgg caccactgca
     2101 ctccagcctg ggcgacagag cgagactgtg tctcaaaaaa aaaaaaaaaa agaagttatt
     2161 gccgtagaga tgtcctagct tccaggtagg gtgcagatga ttccagaatg cctgttttga
     2221 ttcctgatag tctaactgga aacatagttc taattcttta tttttttact ccatcttatc
     2281 ccaggtgtgg tggctatgta gaaaggcaaa atttctgttc attgataaaa aggttaaggg
     2341 ttaaaaggag taggcagtga ctaggagctt tattttaaga ccagcattcc ctgaattggc
     2401 agtttctttg tctccactgg ttccattatt gtctgtgatg actaggggtc atttacagag
     2461 gtagggtttt taaaaggaga gcctttctga tgccactttt ctgcttgaca ggccggacag
     2521 tggctgtgga ttgggccgtg gcaaaggata aatataaaga tacacagtct gtttctgcta
     2581 taggcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     2641 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2701 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2761 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2821 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2881 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2941 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3001 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3061 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3121 gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat
     3181 aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
     3241 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     3301 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     3361 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
     3421 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca
     3481 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
     3541 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
     3601 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     3661 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     3721 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     3781 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     3841 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     3901 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     3961 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     4021 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     4081 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     4141 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     4201 tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga
     4261 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     4321 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     4381 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4441 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     4501 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4561 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4621 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4681 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4741 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4801 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4861 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4921 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4981 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5041 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5101 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5161 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5221 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5281 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5341 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     5401 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5461 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5521 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5581 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5641 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5701 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5761 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5821 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5881 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     5941 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6001 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6061 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6121 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6181 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6241 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6301 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6361 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6421 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6481 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6541 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6601 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6661 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     6721 acaatctgct ctgatgccgc atagttaagc cagta
//
