LOCUS       sequence-333247-        6756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian vector for constitutive expression of RBM28 Exon 5 WT 
            minigene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6756)
  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 6756)
  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 WT minigene
FEATURES             Location/Qualifiers
     source          1..6756
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        114..493
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        494..697
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     648..668
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    699..717
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    720..738
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     748..772
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(2587..2604)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2593..2817
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2950..2969)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3101..3148
                     /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             3156..4178
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3156..4178
                     /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    4308..4429
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4345..4364)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4399..4418
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4478..4494)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4478..4494)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4491..4513)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4502..4518
                     /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(4526..4532,4533..4550,4551..4556))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4571..4592
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4709..4726)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4880..5468)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4960..4979)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5639..6430,6431..6499))
                     /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     6262..6281
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6500..6604)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6679..6698)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
       61 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
      121 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      181 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      241 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      301 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      361 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      421 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      481 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      541 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
      601 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
      661 taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg atagagatct
      721 ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga tcgcctggag
      781 acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccggac
      841 tctagcgttt aaacttaagc ttggtacctg ttcagaagat gacttgaaga cagtatttgc
      901 tcaatttgga gctgtcctgg aagtaaatat ccctaggaaa ccaggtacta ataaaccttc
      961 acttggtgtg gattacgtta gttaacatat tacccttgaa caaacatata tgatatatat
     1021 gaagtatttg gcacagtgct tcacatctaa tgaaaaccca aataatggtt ttattcccct
     1081 gatgatattt tctgttggat cagaattagt tttagaaggc gagttagttt gagaaagtgt
     1141 tttgctctcc ccaagtccaa gtcagagtat actcagtaag aagatgtaag caattgcttt
     1201 ggggaagggc ctgacctgct ggcagaattt gggccatctt gtgggcctga attgcagtaa
     1261 tttacttctt agtattctgg ctacctcctg ctgcctctca cttctttctt tgtgcatttg
     1321 cacacagatg ggaagatgcg cggttttggt tttgttcagt tcaaaaacct cctagaagca
     1381 ggtaaagctc tcaaaggcat gaacatgaaa gagataaaag gtaagctttc tatacccata
     1441 tcattgaccc agatatttct ggtgttttgg tgaattagtg tagtaaccca ctttctgcat
     1501 tttcaaaaga tgtttgcatt attaagagtc tcattgctaa tacggtagct atttaggtga
     1561 aatactctag aacttgatct tggggttaaa cttgaaagaa cccttgccag tattctttct
     1621 tttatctggt gagagtgtcc ttttaaatcc ctagtgaaaa atgtccctga gataattgct
     1681 gatagatgca ggttggtatc tagattcctc cttggttata tcactgttca gcatctgtgg
     1741 gctatttatt aaactgactg tgatttatat attgacttat caagtactgt gcctaaagaa
     1801 gttattgccg gccaggtgcg gtggctcaca cctgtaatcc tagcactttg ggaggccgag
     1861 gaggcaggtg gatcacctga ggtcaggagt tcaagaccag cctggtcaac atggcgaaac
     1921 cccgtctcta ctgaaaatac aaaaattagc caggtgtggt ggtgcatgct tgtaatccca
     1981 gctactcaag aggctgaggc aggagaatcg cttgaacctg gaggcggagg ttgcagtgaa
     2041 ctgagatggc accactgcac tccagcctgg gcgacagagc gagactgtgt ctcaaaaaaa
     2101 aaaaaaaaaa gaagttattg ccgtagagat gtcctagctt ccaggtaggg tgcagatgat
     2161 tccagaatgc ctgttttgat tcctgatagt ctaactggaa acatagttct aattctttat
     2221 ttttttactc catcttatcc caggtgtggt ggctatgtag aaaggcaaaa tttctgttca
     2281 ttgataaaaa ggttaagggt taaaaggagt aggcagtgac taggagcttt attttaagac
     2341 cagcattccc tgaattggca gtttctttgt ctccactggt tccattattg tctgtgatga
     2401 ctaggggtca tttacagagg tagggttttt aaaaggagag cctttctgat gccacttttc
     2461 tgcttgacag gccggacagt ggctgtggat tgggccgtgg caaaggataa atataaagat
     2521 acacagtctg tttctgctat aggcggccgc tcgagtctag agggcccgtt taaacccgct
     2581 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2641 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     2701 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     2761 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     2821 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     2881 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2941 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3001 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3061 accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt
     3121 cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt
     3181 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg
     3241 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg
     3301 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg
     3361 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt
     3421 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg
     3481 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga
     3541 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt
     3601 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg
     3661 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg
     3721 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc
     3781 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg
     3841 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg
     3901 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg
     3961 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc
     4021 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg
     4081 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa
     4141 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt
     4201 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4261 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4321 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4381 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4441 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4501 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4561 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4621 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4681 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4741 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4801 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4861 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4921 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4981 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5041 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5101 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5161 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5221 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5281 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5341 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5401 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5461 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5521 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5581 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5641 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5701 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5761 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5821 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5881 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5941 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6001 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6061 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6121 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6181 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6241 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6301 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6361 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6421 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6481 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6541 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6601 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     6661 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     6721 ccctgcttgt gtgttggagg tcgctgagta gtgcgc
//
