LOCUS       sequence-490914-        5743 bp    DNA     circular SYN 28-OCT-2025
DEFINITION  Expresses R-IscB with EGFP-repairing omega-RNA in mammalian cells 
            for trans-splicing assessments..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5743)
  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 5743)
  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-NLS_T2A_mCherry_U1a-...
FEATURES             Location/Qualifiers
     source          1..5743
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      333..342
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(390..411)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    874..929
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(928..947)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    958..1182
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1171..1188
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1220..1930)
                     /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(1498..1517)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1767..1785
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1931..1984)
                     /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(1988..2035)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(3371..3391)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      3394..3403
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3428..3631)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3457..3477)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3632..4011
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4019..4123
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4124..4192,4193..4984)
                     /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(4342..4361)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5155..5743
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5644..5663
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggcatgcaag cttgggcggc ctctagaatg gaggcggtac tatgtagatg agaattcagg
       61 agcaaactgg gaaaagcaac tgcttccaaa tatttgtgat ttttacagtg tagttttgga
      121 aaaactctta gcctaccaat tcttctaagt gttttaaaat gtgggagcca gtacacatga
      181 agttatagag tgttttaatg aggcttaaat atttaccgta actatgaaat gctacgcata
      241 tcatgctgtt caggctccgt ggccacgcaa ctcatactta agcagacagt ggttcaaagt
      301 ttttttcttc catttcaggt gtcgtgaaca ccgccaccat ggtgatggtg agcaagggcg
      361 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
      421 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
      481 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
      541 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
      601 agtccgccat gcccgaaggc tacgtccagg taagagagct cgttgcgata ttatgagaga
      661 acattcttgc aggaaaaatg agggggctct ttcaacttga aaaggttgaa agagcacagg
      721 ctgagacatt cgtaaggccg aaaggccgga cgcaccctgg gatttcccca gtccccggaa
      781 ctgcatagcg gatgtcagtt gatgaaaatc agataagcca gggggaacaa tcacctctct
      841 ggaaacagag agagtttttt ttgttttgca gtaaataaag gaaatttatt ttcattgcaa
      901 tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatccca
      961 tagagcccac cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt
     1021 cctgccccac cccacccccc agaatagaat gacacctact cagacaatgc gatgcaattt
     1081 cctcatttta ttaggaaagg acagtgggag tggcaccttc cagggtcaag gaaggcacgg
     1141 gggaggggca aacaacagat ggctggcaac tagaaggcac agtcgaggct gatcagcggg
     1201 tttaaacggg ccctctagat tacttgtaca gctcgtccat gccgccggtg gagtggcggc
     1261 cctcggcgcg ttcgtactgt tccacgatgg tgtagtcctc gttgtgggag gtgatgtcca
     1321 acttgatgtt gacgttgtag gcgccgggca gctgcacggg cttcctggcc ttgtaggtgg
     1381 tcttgacctc agcgtcgtag tggccgccgt ccttcagctt cagcctctgc ttgatctcgc
     1441 ccttcagggc gccgtcctcg gggtacatcc gctcggagga ggcctcccag cccatggtct
     1501 tcttctgcat tacggggccg tcggagggga agttggtgcc gcgcagcttc accttgtaga
     1561 tgaactcgcc gtcctgcagg gaggagtcct gggtcacggt caccacgccg ccgtcctcga
     1621 agttcatcac gcgctcccac ttgaagccct cggggaagga cagcttcaag tagtcgggga
     1681 tgtcggcggg gtgcttcacg taggccttgg agccgtacat gaactgaggg gacaggatgt
     1741 cccaggcgaa gggcaggggg ccacccttgg tcaccttcag cttggcggtc tgggtgccct
     1801 cgtaggggcg gccctcgccc tcgccctcga tctcgaactc gtggccgttc acggagccct
     1861 ccatgtgcac cttgaagcgc atgaactcct tgatgatggc catgttatcc tcctcgccct
     1921 tgctcaccat tgggccggga ttttcctcca cgtccccgca tgttagaaga cttcccctgc
     1981 cctctccctt tttctttttt gcctggccgg cctttttcgt ggccgccggc cttttggacc
     2041 ccacgtagat ctgcagtccg ccgttgtact gagcagaagg gtgcttcacg gtcagtttag
     2101 acacgtcagc ggcgctgtga gcggctctgt actcttccag gctgtcagtt ttctggtcca
     2161 tagctttgtg ccggttggtg gcaactttct tgccgcccat gtagtagctc cggttcagat
     2221 tggccttgtg acaggcctgt ctatcatgcc ttctgaactg gtgcaccatg tatggtctgc
     2281 ccttagggct gctcaccttc ttggcgtcgg tcagtgcgct gcaggcgatg cagtaggcgt
     2341 ccaggtagtg gtccttaggg atgccatgct cttcccgaaa cagataggtg tcttggccgc
     2401 ttgtcacgca gaaatttccg gggaacatat cggccagctg atcagccagg taggggatga
     2461 tctggttcag cacagacagg gcgtggtatt tcttgttcat tccagacttc ttgctggcca
     2521 ggttggcctc ccactcctta tctgtgtgca ccagtctcgc gtgttcttca caaaggccca
     2581 ctctgttttc cagtgtctcg ctgccgttct ttcttctggg gaccacgtgg tggtagtggt
     2641 cgatgccgtg tttacaaaac aggcagtgtc catcctgctg catggacacg gcctcttcca
     2701 cgctgccttt gccgtacaga gggccccgct ggtactgcca tctctgaacc ttggggttgt
     2761 tcatggccat aaagctgaac cgattcagct ccagcaccac cttggccacg ggcaggatct
     2821 tctgcacctt cttaaccacg ttcaggtggg tgaccagcag gtgattggcg gtaggtgtca
     2881 gccatccttt aggccgcttg cggttgttga atctggcttc cttgttccgg atgctcttgc
     2941 atgtaatagg ctttttgcag ccaggcagca gtctttgctt ttcgccctcc tcgaaggcgg
     3001 tgccggcagc cttggctctc cgccgtcttt tgcaccgccg cttcagcctt ctgtgggcac
     3061 gcctatactg ctttctgtcc ttcatcagct taggcacgtc cttgtttctt gtctcgattt
     3121 gggcgttgaa cacgctctct ccgctctcgg tcaccacgct cataccgata ttggttctgc
     3181 caggagcaat gcccagcacc agaggctggg tttcctcggc agactcgtag gtcagctgga
     3241 tggtgaaggg ctttctttcc accactctag ccttgccttc cttgagcaag atccgcacgt
     3301 ggccgcatct ggtggtgggc atcagtggtt tgccggactt agagatcacg tagaccacgg
     3361 ccatagatcc caccttccgt ttcttttttg gtcccatggt ggcagtgggt tctctagtta
     3421 gccagagagc tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca
     3481 atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac
     3541 tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac
     3601 gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga
     3661 tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc
     3721 atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg
     3781 ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg
     3841 gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc
     3901 agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa
     3961 ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt caaatgtgcg
     4021 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4081 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     4141 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4201 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     4261 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     4321 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     4381 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     4441 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     4501 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     4561 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     4621 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     4681 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     4741 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     4801 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     4861 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     4921 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     4981 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     5041 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     5101 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5161 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5221 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5281 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     5341 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5401 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5461 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5521 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5581 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5641 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5701 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaa
//
