LOCUS       sequence-490873-        5340 bp    DNA     circular SYN 28-OCT-2025
DEFINITION  All-in-one plasmid. Expresses R-IscB in mammalian cells. Dead 
            mutations 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 Oct 28, 2025 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-dead R-IscB (D60A,
            H269A)-NLS_T2...
FEATURES             Location/Qualifiers
     source          1..5340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(16..604)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(96..115)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(775..1566,1567..1635))
                     /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     1398..1417
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1636..1740)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1748..2127
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2128..2331
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2282..2302
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2356..2365
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2368..2388
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3724..3771
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3775..3828
                     /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             3829..4539
                     /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(3974..3992)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4242..4261
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4571..4588)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4577..4801
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4812..4831
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    4830..4885
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     promoter        complement(5092..5340)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(5150..5169)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5320..5340)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 ccaagcttgc atgcctttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
       61 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      121 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      181 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      241 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      301 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      361 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      421 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
      481 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
      541 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
      601 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
      661 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
      721 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
      781 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
      841 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
      901 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
      961 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     1021 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     1081 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     1141 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     1201 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     1261 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     1321 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     1381 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     1441 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     1501 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     1561 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     1621 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     1681 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     1741 cacatttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
     1801 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     1861 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     1921 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     1981 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     2041 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     2101 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
     2161 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
     2221 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
     2281 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta
     2341 actagagaac ccactgccac catgggacca aaaaagaaac ggaaggtggg atctatggcc
     2401 gtggtctacg tgatctctaa gtccggcaaa ccactgatgc ccaccaccag atgcggccac
     2461 gtgcggatct tgctcaagga aggcaaggct agagtggtgg aaagaaagcc cttcaccatc
     2521 cagctgacct acgagtctgc cgaggaaacc cagcctctgg tgctgggcat tgctcctggc
     2581 agaaccaata tcggtatgag cgtggtgacc gagagcggag agagcgtgtt caacgcccaa
     2641 atcgagacaa gaaacaagga cgtgcctaag ctgatgaagg acagaaagca gtataggcgt
     2701 gcccacagaa ggctgaagcg gcggtgcaaa agacggcgga gagccaaggc tgccggcacc
     2761 gccttcgagg agggcgaaaa gcaaagactg ctgcctggct gcaaaaagcc tattacatgc
     2821 aagagcatcc ggaacaagga agccagattc aacaaccgca agcggcctaa aggatggctg
     2881 acacctaccg ccaatcacct gctggtcacc cacctgaacg tggttaagaa ggtgcagaag
     2941 atcctgcccg tggccaaggt ggtgctggag ctgaatcggt tcagctttat ggccatgaac
     3001 aaccccaagg ttcagagatg gcagtaccag cggggccctc tgtacggcaa aggcagcgtg
     3061 gaagaggccg tgtccatgca gcaggatgga cactgcctgt tttgtaaaca cggcatcgac
     3121 cactaccacc acgtggtccc cagaagaaag aacggcagcg agacactgga aaacagagtg
     3181 ggcctttgtg aagaacacgc gagactggtg cacacagata aggagtggga ggccaacctg
     3241 gccagcaaga agtctggaat gaacaagaaa taccacgccc tgtctgtgct gaaccagatc
     3301 atcccctacc tggctgatca gctggccgat atgttccccg gaaatttctg cgtgacaagc
     3361 ggccaagaca cctatctgtt tcgggaagag catggcatcc ctaaggacca ctacctggac
     3421 gcctactgca tcgcctgcag cgcactgacc gacgccaaga aggtgagcag ccctaagggc
     3481 agaccataca tggtgcacca gttcagaagg catgatagac aggcctgtca caaggccaat
     3541 ctgaaccgga gctactacat gggcggcaag aaagttgcca ccaaccggca caaagctatg
     3601 gaccagaaaa ctgacagcct ggaagagtac agagccgctc acagcgccgc tgacgtgtct
     3661 aaactgaccg tgaagcaccc ttctgctcag tacaacggcg gactgcagat ctacgtgggg
     3721 tccaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gggagagggc
     3781 aggggaagtc ttctaacatg cggggacgtg gaggaaaatc ccggcccaat ggtgagcaag
     3841 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     3901 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     3961 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     4021 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     4081 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     4141 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     4201 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     4261 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     4321 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     4381 acctacaagg ccaggaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     4441 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     4501 cgccactcca ccggcggcat ggacgagctg tacaagtaat ctagagggcc cgtttaaacc
     4561 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     4621 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     4681 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     4741 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     4801 ggatttgccc tcccatatgt ccttccgagt gagagacaca aaaaattcca acacactatt
     4861 gcaatgaaaa taaatttcct ttatttactg caaaacaaaa aaaactctct ctgtttccag
     4921 agaggtgatt gttccccctg gcttatctga ttttcatcaa ctgacatccg ctatgcagtt
     4981 ccggggactg gggaaatccc agggtgcgtc cggcctttcg gccttacgaa tgtctcagcc
     5041 tgtgctcttt caaccttttc aagttgaaag agccctgcgc caaggtgcgg cggtgtttcg
     5101 tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat
     5161 atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt
     5221 tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttccaattat
     5281 ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc
//
