LOCUS       sequence-333100-        7835 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian vector for constitutive expression of RBM28 Exon 8 G>T 
            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 G>T minigene
FEATURES             Location/Qualifiers
     source          1..7835
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        129..508
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        509..712
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     663..683
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    714..732
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    735..753
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     763..787
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     RBS             1216..1224
                     /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(3681..3698)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3687..3911
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4044..4063)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4195..4242
                     /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             4250..5272
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4250..5272
                     /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    5402..5523
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5439..5458)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5493..5512
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5572..5588)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5572..5588)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5585..5607)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5596..5612
                     /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(5620..5626,5627..5644,5645..5650))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5665..5686
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5803..5820)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5974..6562)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6054..6073)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6733..7524,7525..7593))
                     /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     7356..7375
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7594..7698)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7773..7792)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
       61 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
      121 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
      181 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      241 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      301 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      361 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      421 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      481 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      541 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      601 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      661 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctccctat
      721 cagtgataga gatctcccta tcagtgatag agatcgtcga cgagctcgtt tagtgaaccg
      781 tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg
      841 atccagcctc cggactctag cgtttaaact taagcttggt accgtgagga aaagagccat
      901 gaatctaaac atcaggaatc agttaaaaag aagggcagag aggaagagga tatggaagag
      961 gaagaaaacg atgatgatga cgatgatgat gatgaagaag atggggtttt tgatgatgaa
     1021 gatgaagagg aagagaatat agaatcaaag gtgaccaagc ctgtgcaaat tcagaagagg
     1081 taagcagctc atcctgttct gagacagcag aggaagattc aagattgtct ccgagaggaa
     1141 aaattctcct ttttctgcct gcatcattgc agaattgaca tgtgtaatga aattgaatag
     1201 gccctctgtc agcccacctc cttagttctg atctgtgcca ttgcagagca gtcaagagac
     1261 cagcccctgc aaaaagcagt gatcattctg aggaggacag tgacctagag gaaagcgata
     1321 gtattgatga tggagaggaa ctggctcaga gtgataccag cactgaggag caagaggata
     1381 aatgtttgtt tggattttca taaatgtctt tagactttaa aaaagttact gaagttacgg
     1441 aaatgagcat cagttctgct tagatctctt tttaaatagg actctaaggt gttctatcaa
     1501 atttatgttt aaatgctgaa aaggaaacct ttatactcct ttcttccaaa ggtttttttg
     1561 ttgtttcctt tttgtttgtt ttgtttttta agtgagtttt ggagtggtgt ttgagggtta
     1621 agattcttag ggaattgaaa acctagctta atgctgaggt tctacctctc tctggattgt
     1681 ccaacttgat agaataacca ggtaaacagc atcttataca gataatatag atgaaacaga
     1741 tgatataacc atctcaacat caagctagat tttaaacaac tccttggagt tgagatgggg
     1801 ataaagaact gtttagtcag tttggactga cataacaaaa taccataaac tgggtggctt
     1861 aaacagcaga aacttatttt ctcacagctc tagaggctag aagtcccagg tgccagctga
     1921 ttcattttgg gtgcagctct cttcctagct tgcagactgc tgccttctcg ctatgtgctt
     1981 gcatggcctt ttctttgtgg cttgacgagg ggagagagag aacaagtaac agtgaactct
     2041 ggtctctctt ttcttataag cacagcaatc ctatcagatt agggcccaac cttaagacct
     2101 catttagcta gcctcagtta cctccctaaa gtctctgtct ccaaatacaa ttatatttgg
     2161 ggttagggct tcaatgtatg aattttgggg gaacacagtg cgttccataa caaggacctc
     2221 tttgaacatg ttgtaaaact ttagccaatc cttattgcta cctttattga gaataaattc
     2281 aacttgttta ttgaggaagg caggtcatta cagaatgaga aatggctaat aactggttct
     2341 ttgatttgtc tgggtggaga tttttatttc agctgttttt attctgattc catttcagta
     2401 agtctgacat gttggtttga ccatataaga aatctgagga aataagcttt ttctgtgagg
     2461 agagaatggt atacacctta gaatcaaaga agcttctaca ctgaagggac tctgagatcc
     2521 tgtattctac aacccacagt cagctgtctc atgccattct ttcacctaaa aaacttatag
     2581 gacagataaa tgataatgat aaaaagttaa aacacaacac agaagagtgt aatacacaaa
     2641 gtagaagttt ctcaccaatc ctaacaattc cctgaagtaa tcactgttgt ttcttgtgca
     2701 tccttccaga tattttcttt gtatgtataa gcttatatgt ctgttcttga aaacaaacag
     2761 tgagtctccc ttatacccag atcctcagct tcttagggaa cttccccaga gacaaccaag
     2821 gttatttgtt tcttgtgtat ctatagatat tttaatacat ttataagcaa aatgcacaca
     2881 tatacacaca catatacaca ttcaaaatgt agtgtatttt ataatttgtg tgtgattgta
     2941 tgtgtatgtt ttttcctcaa agtttcctat gtcctataca gtatttccag aaagtttaca
     3001 ctgtcatagt agtatatgga aatgtctttt tcccataacc ttaccacatt ggattccatc
     3061 agtctttttg attttgcaat ctgttggtga aaaatagtat ctcatttctt gtgctatgtt
     3121 ttctttaatt aagagtgagt ttgaacatct tttcatatac ttagcatgtc ctttgtctat
     3181 ttttattttt atttatttat tttttttgag actttgtttc gctcttgtca ccgtggctgg
     3241 agtgcaatgg ctcaatcttg gctcactgca acctccacct cccaggttca agggattctc
     3301 ctgcctcagc ctcctgagta gctgggacta taggtgcctg ctaccacgcc tggctaattt
     3361 ttgtattttt agtagagaca gggttttgcc atgttggcca ggctggtctc gaactcctga
     3421 cctcaggtga tcagcccgcc tgggcttttc aaagtggcat gagccactgc gccaggctta
     3481 tctttatgta cttaagaagt tagctctttg tcatgtggct tacactaatc tctttcagtt
     3541 tgttgttttt ctttttacct tagctgtgca agtctcaaac aaaaagaaga ggaaattacc
     3601 ctctgatgtg aatgaaggga aaactgtttt tatcaggcgg ccgctcgagt ctagagggcc
     3661 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3721 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3781 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3841 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3901 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     3961 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4021 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4081 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4141 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta cctagaagtt
     4201 cctattccga agttcctatt ctctagaaag tataggaact tccttggcca aaaagcctga
     4261 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct
     4321 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg
     4381 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg
     4441 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga
     4501 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga
     4561 aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc
     4621 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac
     4681 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt
     4741 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc
     4801 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct
     4861 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc
     4921 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca
     4981 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta
     5041 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga
     5101 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg
     5161 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact
     5221 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agcacgtact
     5281 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     5341 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     5401 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     5461 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5521 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     5581 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5641 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5701 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5761 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5821 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5881 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5941 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     6001 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     6061 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6121 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     6181 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6241 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6301 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6361 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6421 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6481 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6541 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6601 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6661 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6721 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6781 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6841 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6901 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6961 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7021 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7081 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7141 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7201 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7261 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7321 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7381 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7441 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7501 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7561 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7621 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7681 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     7741 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     7801 agccagtatc tgctccctgc ttgtgtgttg gaggt
//
