LOCUS       sequence-333248-        7835 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian vector for constitutive expression of RBM28 Exon 8 WT 
            minigene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7835)
  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 7835)
  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 E8 WT minigene
FEATURES             Location/Qualifiers
     source          1..7835
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        89..468
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        469..672
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     623..643
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    674..692
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    695..713
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     723..747
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     RBS             1176..1184
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(3641..3658)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3647..3871
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4004..4023)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4155..4202
                     /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             4210..5232
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4210..5232
                     /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    5362..5483
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5399..5418)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5453..5472
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5532..5548)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5532..5548)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5545..5567)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5556..5572
                     /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(5580..5586,5587..5604,5605..5610))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5625..5646
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5763..5780)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5934..6522)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6014..6033)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6693..7484,7485..7553))
                     /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     7316..7335
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7554..7658)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7733..7752)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
       61 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
      121 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      181 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      241 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      301 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      361 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      421 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      481 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      541 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      601 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      661 ataagcagag ctctccctat cagtgataga gatctcccta tcagtgatag agatcgtcga
      721 cgagctcgtt tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc
      781 catagaagac accgggaccg atccagcctc cggactctag cgtttaaact taagcttggt
      841 accgtgagga aaagagccat gaatctaaac atcaggaatc agttaaaaag aagggcagag
      901 aggaagagga tatggaagag gaagaaaacg atgatgatga cgatgatgat gatgaagaag
      961 atggggtttt tgatgatgaa gatgaagagg aagagaatat agaatcaaag gtgaccaagc
     1021 ctgtgcaaat tcagaagagg taagcagctc atcctgttct gagacagcag aggaagattc
     1081 aagattgtct ccgagaggaa aaattctcct ttttctgcct gcatcattgc agaattgaca
     1141 tgtgtaatga aattgaatag gccctctgtc agcccacctc cttagttctg atctgtgcca
     1201 ttgcagagca gtcaagagac cagcccctgc aaaaagcagt gatcattctg aggaggacag
     1261 tgacctagag gaaagcgata gtattgatga tggagaggaa ctggctcaga gtgataccag
     1321 cactgaggag caagaggata aaggtttgtt tggattttca taaatgtctt tagactttaa
     1381 aaaagttact gaagttacgg aaatgagcat cagttctgct tagatctctt tttaaatagg
     1441 actctaaggt gttctatcaa atttatgttt aaatgctgaa aaggaaacct ttatactcct
     1501 ttcttccaaa ggtttttttg ttgtttcctt tttgtttgtt ttgtttttta agtgagtttt
     1561 ggagtggtgt ttgagggtta agattcttag ggaattgaaa acctagctta atgctgaggt
     1621 tctacctctc tctggattgt ccaacttgat agaataacca ggtaaacagc atcttataca
     1681 gataatatag atgaaacaga tgatataacc atctcaacat caagctagat tttaaacaac
     1741 tccttggagt tgagatgggg ataaagaact gtttagtcag tttggactga cataacaaaa
     1801 taccataaac tgggtggctt aaacagcaga aacttatttt ctcacagctc tagaggctag
     1861 aagtcccagg tgccagctga ttcattttgg gtgcagctct cttcctagct tgcagactgc
     1921 tgccttctcg ctatgtgctt gcatggcctt ttctttgtgg cttgacgagg ggagagagag
     1981 aacaagtaac agtgaactct ggtctctctt ttcttataag cacagcaatc ctatcagatt
     2041 agggcccaac cttaagacct catttagcta gcctcagtta cctccctaaa gtctctgtct
     2101 ccaaatacaa ttatatttgg ggttagggct tcaatgtatg aattttgggg gaacacagtg
     2161 cgttccataa caaggacctc tttgaacatg ttgtaaaact ttagccaatc cttattgcta
     2221 cctttattga gaataaattc aacttgttta ttgaggaagg caggtcatta cagaatgaga
     2281 aatggctaat aactggttct ttgatttgtc tgggtggaga tttttatttc agctgttttt
     2341 attctgattc catttcagta agtctgacat gttggtttga ccatataaga aatctgagga
     2401 aataagcttt ttctgtgagg agagaatggt atacacctta gaatcaaaga agcttctaca
     2461 ctgaagggac tctgagatcc tgtattctac aacccacagt cagctgtctc atgccattct
     2521 ttcacctaaa aaacttatag gacagataaa tgataatgat aaaaagttaa aacacaacac
     2581 agaagagtgt aatacacaaa gtagaagttt ctcaccaatc ctaacaattc cctgaagtaa
     2641 tcactgttgt ttcttgtgca tccttccaga tattttcttt gtatgtataa gcttatatgt
     2701 ctgttcttga aaacaaacag tgagtctccc ttatacccag atcctcagct tcttagggaa
     2761 cttccccaga gacaaccaag gttatttgtt tcttgtgtat ctatagatat tttaatacat
     2821 ttataagcaa aatgcacaca tatacacaca catatacaca ttcaaaatgt agtgtatttt
     2881 ataatttgtg tgtgattgta tgtgtatgtt ttttcctcaa agtttcctat gtcctataca
     2941 gtatttccag aaagtttaca ctgtcatagt agtatatgga aatgtctttt tcccataacc
     3001 ttaccacatt ggattccatc agtctttttg attttgcaat ctgttggtga aaaatagtat
     3061 ctcatttctt gtgctatgtt ttctttaatt aagagtgagt ttgaacatct tttcatatac
     3121 ttagcatgtc ctttgtctat ttttattttt atttatttat tttttttgag actttgtttc
     3181 gctcttgtca ccgtggctgg agtgcaatgg ctcaatcttg gctcactgca acctccacct
     3241 cccaggttca agggattctc ctgcctcagc ctcctgagta gctgggacta taggtgcctg
     3301 ctaccacgcc tggctaattt ttgtattttt agtagagaca gggttttgcc atgttggcca
     3361 ggctggtctc gaactcctga cctcaggtga tcagcccgcc tgggcttttc aaagtggcat
     3421 gagccactgc gccaggctta tctttatgta cttaagaagt tagctctttg tcatgtggct
     3481 tacactaatc tctttcagtt tgttgttttt ctttttacct tagctgtgca agtctcaaac
     3541 aaaaagaaga ggaaattacc ctctgatgtg aatgaaggga aaactgtttt tatcaggcgg
     3601 ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     3661 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     3721 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     3781 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     3841 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     3901 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3961 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4021 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4081 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     4141 ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact
     4201 tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag
     4261 ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc
     4321 ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac
     4381 aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt
     4441 gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc
     4501 acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc
     4561 atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg
     4621 caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat
     4681 gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc
     4741 gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat
     4801 ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc
     4861 gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg
     4921 ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga
     4981 tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg
     5041 gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga
     5101 tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc
     5161 gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg
     5221 gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg
     5281 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     5341 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     5401 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5461 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     5521 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5581 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5641 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5701 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5761 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5821 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5881 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5941 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     6001 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6061 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6121 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6181 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6241 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6301 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6361 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6421 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6481 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6541 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6601 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6661 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6721 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6781 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6841 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6901 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6961 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     7021 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7081 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7141 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7201 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7261 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7321 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7381 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7441 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7501 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7561 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7621 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7681 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     7741 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     7801 agtagtgcgc gagcaaaatt taagctacaa caagg
//
