LOCUS       sequence-488935-        5829 bp    DNA     circular SYN 09-OCT-2025
DEFINITION  Expresses inactive EGFP (Y67X) in mammalian cells for trans-splicing
            assessments. LMNA intron is inserted into EGFP CDS..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5829)
  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 5829)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 9, 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_EGFP (Y67X)_...
FEATURES             Location/Qualifiers
     source          1..5829
                     /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"
     primer_bind     complement(3874..3895)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4872..4891)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5219..5240
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5317..5334)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5323..5547
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5558..5577
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        complement(5581..5829)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(5639..5658)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5809..5829)
                     /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 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     3901 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     3961 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     4021 ctgacctaag gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     4081 ttcaagtccg ccatgcccga aggctacgtc caggtagccg ccgctgaggc cgagcctgca
     4141 ctggggccac ccagccaggc ctgggggcag cctctcccca gcctccccgt gccaaaaatc
     4201 ttttcattaa agaatgtttt ggaactttac tcgctggcct ggcctttctt ctctctcctc
     4261 cctatacctt gaacagggaa cccaggtgtc tgggtgccct actctggtaa ggaagggagt
     4321 gggaactttc tgatgccatg gaatattcct gtgggagcag tggacaaggg tctggatttg
     4381 tcttctggga aagggagggg aggacagacg tggggcatgc ccgccctgcc tctctccccc
     4441 attcttgttg catgcatatc ctctcatttc cctcattttt cctgcaagaa tgttctctct
     4501 cattcctgac cgcccctcca ctccaattaa tagtgcatgc ctgctgccct acaagcttgc
     4561 tcccgttctc tcttcttttc ctcttaagct cagagtagct agaacagagt cagagtcact
     4621 gctctggttc tctgtcccca agtcttcctg agccttctcc ccttttatgt cttccctctc
     4681 ctcctccggg cccctagcct cccaaacccc cattgcccgc tggctccttg ggcacagaac
     4741 cacaccttcc tgcctggcgg ctgggagcct gcaggagcct ggagcctggt tgggcctgag
     4801 tggtcagtcc cagactcgcc gtcccgcctg agccttgtct cccttcccag gagcgcacca
     4861 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     4921 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     4981 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     5041 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     5101 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     5161 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     5221 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     5281 agtaatctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     5341 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     5401 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     5461 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     5521 atgctgggga tgcggtgggc tctatgggat ttgccctccc atatgtcctt ccgagtgagc
     5581 ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac
     5641 ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa
     5701 ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat
     5761 tccaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa
     5821 taggccctc
//
