LOCUS       sequence-496263-        5340 bp    DNA     circular SYN 06-JAN-2026
DEFINITION  All-in-one plasmid. Expresses R-IscB in mammalian cells. ssRNase 
            enhancing mutations (H243E, H269N, R270Q) included..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5340)
  AUTHORS   Xu C, Niu X, Sun H, Yan H, Tang W, Ke A
  TITLE     Conversion of IscB and Cas9 into RNA-guided RNA editors.
  JOURNAL   Cell. 2025 Aug 18:S0092-8674(25)00854-2. doi: 
            10.1016/j.cell.2025.07.032.
  PUBMED    40829585
REFERENCE   2  (bases 1 to 5340)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 6, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2025 
            Aug 18:S0092-8674(25)00854-2. doi: 10.1016/j.cell.2025.07.032."
COMMENT     Sequence Label: pMini-CMV-NLS-R-IscB-VR4-NLS (D60A, H243E, H26...
FEATURES             Location/Qualifiers
     source          1..5340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     polyA_signal    456..511
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(510..529)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    540..764
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     753..770
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(802..1512)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKARKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1080..1099)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1349..1367
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1513..1566)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(1570..1617)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(2953..2973)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      2976..2985
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3010..3213)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3039..3059)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3214..3593
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3601..3705
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3706..3774,3775..4566)
                     /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     complement(3924..3943)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4737..5325
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5226..5245
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg catgactgtc acacttggct ctttcaactt gaaaaggttg aaagagcaca
      301 ggctgagaca ttcgtaaggc cgaaaggccg gacgcaccct gggatttccc cagtccccgg
      361 aactgcatag cggatgtcag ttgatgaaaa tcagataagc cagggggaac aatcacctct
      421 ctggaaacag agagagtttt ttttgttttg cagtaaataa aggaaattta ttttcattgc
      481 aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatcc
      541 catagagccc accgcatccc cagcatgcct gctattgtct tcccaatcct cccccttgct
      601 gtcctgcccc accccacccc ccagaataga atgacaccta ctcagacaat gcgatgcaat
      661 ttcctcattt tattaggaaa ggacagtggg agtggcacct tccagggtca aggaaggcac
      721 gggggagggg caaacaacag atggctggca actagaaggc acagtcgagg ctgatcagcg
      781 ggtttaaacg ggccctctag attacttgta cagctcgtcc atgccgccgg tggagtggcg
      841 gccctcggcg cgttcgtact gttccacgat ggtgtagtcc tcgttgtggg aggtgatgtc
      901 caacttgatg ttgacgttgt aggcgccggg cagctgcacg ggcttcctgg ccttgtaggt
      961 ggtcttgacc tcagcgtcgt agtggccgcc gtccttcagc ttcagcctct gcttgatctc
     1021 gcccttcagg gcgccgtcct cggggtacat ccgctcggag gaggcctccc agcccatggt
     1081 cttcttctgc attacggggc cgtcggaggg gaagttggtg ccgcgcagct tcaccttgta
     1141 gatgaactcg ccgtcctgca gggaggagtc ctgggtcacg gtcaccacgc cgccgtcctc
     1201 gaagttcatc acgcgctccc acttgaagcc ctcggggaag gacagcttca agtagtcggg
     1261 gatgtcggcg gggtgcttca cgtaggcctt ggagccgtac atgaactgag gggacaggat
     1321 gtcccaggcg aagggcaggg ggccaccctt ggtcaccttc agcttggcgg tctgggtgcc
     1381 ctcgtagggg cggccctcgc cctcgccctc gatctcgaac tcgtggccgt tcacggagcc
     1441 ctccatgtgc accttgaagc gcatgaactc cttgatgatg gccatgttat cctcctcgcc
     1501 cttgctcacc attgggccgg gattttcctc cacgtccccg catgttagaa gacttcccct
     1561 gccctctccc tttttctttt ttgcctggcc ggcctttttc gtggccgccg gccttttgga
     1621 ccccacgtag atctgcagtc cgccgttgta ctgagcagaa gggtgcttca cggtcagttt
     1681 agacacgtca gcggcgctgt gagcggctct gtactcttcc aggctgtcag ttttctggtc
     1741 catagctttg tgccggttgg tggcaacttt cttgccgccc atgtagtagc tccggttcag
     1801 attggccttg tgacaggcct gtctatcatg ccttctgaac tggtgcacca tgtatggtct
     1861 gcccttaggg ctgctcacct tcttggcgtc ggtcagtgcg ctgcaggcga tgcagtaggc
     1921 gtccaggtag tggtccttag ggatgccatg ctcttcccga aacagatagg tgtcttggcc
     1981 gcttgtcacg cagaaatttc cggggaacat atcggccagc tgatcagcca ggtaggggat
     2041 gatctggttc agcacagaca gggcgtggta tttcttgttc attccagact tcttgctggc
     2101 caggttggcc tcccactcct tatctgtgtg caccagctgg ttgtgttctt cacaaaggcc
     2161 cactctgttt tccagtgtct cgctgccgtt ctttcttctg gggaccacgt ggtggtactc
     2221 gtcgatgccg tgtttacaaa acaggcagtg tccatcctgc tgcatggaca cggcctcttc
     2281 cacgctgcct ttgccgtaca gagggccccg ctggtactgc catctctgaa ccttggggtt
     2341 gttcatggcc ataaagctga accgattcag ctccagcacc accttggcca cgggcaggat
     2401 cttctgcacc ttcttaacca cgttcaggtg ggtgaccagc aggtgattgg cggtaggtgt
     2461 cagccatcct ttaggccgct tgcggttgtt gaatctggct tccttgttcc ggatgctctt
     2521 gcatgtaata ggctttttgc agccaggcag cagtctttgc ttttcgccct cctcgaaggc
     2581 ggtgccggca gccttggctc tccgccgtct tttgcaccgc cgcttcagcc ttctgtgggc
     2641 acgcctatac tgctttctgt ccttcatcag cttaggcacg tccttgtttc ttgtctcgat
     2701 ttgggcgttg aacacgctct ctccgctctc ggtcaccacg ctcataccga tattggttct
     2761 gccaggagca atgcccagca ccagaggctg ggtttcctcg gcagactcgt aggtcagctg
     2821 gatggtgaag ggctttcttt ccaccactct agccttgcct tccttgagca agatccgcac
     2881 gtggccgcat ctggtggtgg gcatcagtgg tttgccggac ttagagatca cgtagaccac
     2941 ggccatagat cccaccttcc gtttcttttt tggtcccatg gtggcagtgg gttctctagt
     3001 tagccagaga gctctgctta tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt
     3061 caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt gccaaaacaa
     3121 actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa accgctatcc
     3181 acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga ctaatacgta
     3241 gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat gccaggcggg
     3301 ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca cttgatgtac
     3361 tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga aagtccctat
     3421 tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt cgttgggcgg
     3481 tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat atgggctatg
     3541 aactaatgac cccgtaattg attactatta ataactagtc aataatcaat gtcaaatgtg
     3601 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3661 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     3721 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     3781 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     3841 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3901 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     3961 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     4021 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     4081 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     4141 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     4201 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     4261 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     4321 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     4381 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     4441 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     4501 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     4561 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     4621 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4681 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4741 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4801 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4861 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     4921 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4981 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5041 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5101 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5161 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5221 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5281 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaggcat gcaagcttgg
//
