LOCUS       sequence-434400-        7071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NMD-reporter plasmid that expresses a a firefly luciferase gene 
            fused to exons 22-27 of the CFTR gene and a synthetic intron 
            (c.3846G>A mutation; W1282X). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7071)
  AUTHORS   Nemudraia A, Nemudryi A, Wiedenheft B
  TITLE     Repair of CRISPR-guided RNA breaks enables site-specific RNA 
            excision in human cells.
  JOURNAL   Science. 2024 Apr 25:eadk5518. doi: 10.1126/science.adk5518.
  PUBMED    38662916
REFERENCE   2  (bases 1 to 7071)
  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: "Science. 
            2024 Apr 25:eadk5518. doi: 10.1126/science.adk5518."
COMMENT     Sequence Label: pAN057
FEATURES             Location/Qualifiers
     source          1..7071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     168..186
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     regulatory      249..258
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(341..544)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(370..390)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        545..848
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(1023..1611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1103..1122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    1940..1987
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2012..2031
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(2219..3013)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(2219..3013)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2330..2349)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2940..2959
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3048..3405)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3062..3197
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        3214..3405
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     3362..3382
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(3407..3511)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3538..3993
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3625..3644)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3835..3856
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4000..4121
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4011..4030)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4065..4084
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4248..4271)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(join(5674..7071,1..252))
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /label=luciferase
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
ORIGIN
        1 gttttcactg catacgacga ttctgtgatt tgtattcagc ccatatcgtt tcatagcttc
       61 tgccaaccga acggacattt cgaagtattc cgcgtacgtg atgttcacct cgatatgtgc
      121 atctgtaaaa gcaattgttc caggaaccag ggcgtatctc ttcatagcct tatgcagttg
      181 ctctccagcg gttccatcct ctagtggata gaatggcgcc gggcctttct ttatgttttt
      241 ggcgtcttcc atggtggcgg taccgtcgac tgcagaattc gaagcttgag ctcgagatct
      301 gagtccggta gcgctagcgg atctgacggt tcactaaacc agctctgctt atatagacct
      361 cccaccgtac acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg
      421 gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac
      481 ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca
      541 tcaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata
      601 aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg
      661 gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact
      721 ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta
      781 ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt
      841 atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt
      901 aataactaat gcatggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      961 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1021 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1081 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1141 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1201 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1261 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1321 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     1381 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     1441 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     1501 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     1561 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     1621 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     1681 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     1741 aaatcaatct aaagtatata tgagtaacct gaggctatgg cagggcctgc cgccccgacg
     1801 ttggctgcga gccctgggcc ttcacccgaa cttggggggt ggggtgggga aaaggaagaa
     1861 acgcgggcgt attggcccca atggggtctc ggtggggtat cgacagagtg ccagccctgg
     1921 gaccgaaccc cgcgtttatg aacaaacgac ccaacaccgt gcgttttatt ctgtcttttt
     1981 attgccgtca tagcgcgggt tccttccggt attgtctcct tccgtgtttc agttagcctc
     2041 cccctagggt gggcgaagaa ctccagcatg agatccccgc gctggaggat catccagccg
     2101 gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt ggaatcgaaa
     2161 tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt cgaaccccag agtcccgctc
     2221 agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac
     2281 cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg
     2341 tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc
     2401 cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga
     2461 cgagatcctc gccgtcgggc atgctcgcct tgagcctggc gaacagttcg gctggcgcga
     2521 gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac
     2581 gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg
     2641 tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag
     2701 atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag
     2761 tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg
     2821 ctgcctcgtc ttgcagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg
     2881 ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg
     2941 cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat
     3001 cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcgatcttt gcaaaagcct
     3061 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga ggcggcctcg
     3121 gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc
     3181 ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga
     3241 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct
     3301 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     3361 caccctaact gacacacatt ccacagctgg ttctttccgc ctcaggactc ttcctttttc
     3421 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     3481 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     3541 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3601 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3661 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3721 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     3781 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3841 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     3901 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3961 cgaattttaa caaaatatta acgcttacaa tttacgcctt aagatacatt gatgagtttg
     4021 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     4081 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     4141 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct
     4201 acaaatgtgg tatggctgat tatgatctag agtcgcggcc gcgattattt atcgtcatcg
     4261 tctttgtagt cgctaccgcc cccaagcctt gtatcttgca cctcttcttc tgtctcctct
     4321 ttcagagcag caatctgggg cttagacttg cacttgcttg agttccggtg gggaaagagc
     4381 ttcaccctgt cggaggggct gatggcttgc cggaagaggc tcctctcgtt cagcagtttc
     4441 tggatggaat cgtactgccg cactttgttc tcttctatga ccaaaaattg ttggcattcc
     4501 agcattgctt ctatcctgtg ttcacagaga attactgtgc aatcagcaaa tgcttgtttt
     4561 agagttcttc taattatttg gtatgttact ggatccaaat gagcactggg ttcatcaagc
     4621 agcaagatct tcgccttact gagaacagat ctagccaagc acatcaactg cttgtggcca
     4681 tggcttagga cacagccccc atccacaagg acaaagtcaa gcttcccagg aaactgttct
     4741 atcacagatc tgagcccaac ctgcagttag agaagatagt tcagatacca cagagttaaa
     4801 aacactcaag cagttgacat tctaaaaata aacacaggag ttctgaaagc ttatgttttt
     4861 cataccaatc cctctacccc cactacctgc caccatttga attgattgag caaaagtcta
     4921 tttacaacct tgacagctca aaagaatcag tctataactt gttacctctt tcaaaatcat
     4981 ttcagttagg ggtaggtcca gtcaaaagta cctgttgctc caggtatgtt agggtactcc
     5041 attttaaagt tgtgcacaca catacatgca cacaaagtgt gtagaatgat gtcagctata
     5101 tcagccattt gtgttggtat gagttacccc tttcaaaatc atttcagtta gcagccttac
     5161 ctcatctgca actttccata tttcttgatc actccactgt tcatagggat ccaagttttt
     5221 tctaaatgtt ccagaaaaaa taaatacttt ctgtggtatc actccaaagg ctttccttca
     5281 ctgttgcaaa gttattgaat cccaagacac accatcgatc tggatttctc cttcagtgtt
     5341 cagtagtctc aaaaaagctg ataacaaagt actcttccct gatccagttc ttcccaagag
     5401 gcccaccctc tggccaggac ttattgagaa ggaaatgttc tctaatatgg catttccacc
     5461 ttctgtgtat tttgctgtga gatctttgac agtcatttgg ccccctgagg gccagatgtc
     5521 atctttcttc acgtgtgaat tctcaataat cataactttc gagagttggc cattcttgta
     5581 tggtttggtt gacttggtag gtttaccttc tgttggcatg tcaatgaact taaagactcg
     5641 gctcacagat cgcatgctac ctccactgcc tcccaatttg gactttccgc ccttcttggc
     5701 ctttatgagg atctctctga tttttcttgc gtcgagtttt ccggtaagac ctttcggtac
     5761 ttcgtccaca aacacaactc ctccgcgcaa ctttttcgcg gttgttactt gactggcgac
     5821 gtaatccacg atctcttttt ccgtcatcgt ctttccgtgc tccaaaacaa caacggcggc
     5881 gggaagttca ccggcgtcat cgtcgggaag acctgccacg cccgcgtcga agatgttggg
     5941 gtgttgtaac aatatcgact ccaattcagc gggggccacc tggtatcctt tgtatttaat
     6001 taaagacttc aagcggtcaa ctatgaagaa gtgttcgtct tcgtcccagt aagctatgtc
     6061 tccagaatgt agccatccat ccttgtcaat caaggcgttg gtcgcttccg gattgtttac
     6121 ataaccggac ataatcatag gtcctctgac acataattcg cctctctgat taacgcccag
     6181 cgttttcccg gtatccagat ccacaacctt cgcttcaaaa aatggaacaa ctttaccgac
     6241 cgcgcccggt ttatcatccc cctcgggtgt aatcagaata gctgatgtag tctcagtgag
     6301 cccatatcct tgtcgtatcc ctggaagatg gaagcgtttt gcaaccgcct ccccgacttc
     6361 tttcgaaaga ggtgcgcccc cagaagcaat ttcgtgtaaa ttagataaat cgtatttgtc
     6421 aatcagagtg cttttggcga agaatgaaaa tagggttggt actagcaacg cactttgaat
     6481 tttgtaatcc tgaagggatc gtaaaaacag ctcttcttca aatctataca ttaagacgac
     6541 tcgaaatcca catatcaaat atccgagtgt agtaaacatt ccaaaaccgt gatggaatgg
     6601 aacaacactt aaaatcgcag tatccggaat gatttgattg ccaaaaatag gatctctggc
     6661 atgcgagaat ctgacgcagg cagttctatg cggaagggcc acacccttag gtaacccagt
     6721 agatccagag gagttcatta tcagtgcaat tgttttgtca cgatcaaagg actctggtac
     6781 aaaatcgtat tcattaaaac cgggaggtag atgagatgtg acgaacgtgt acatcgactg
     6841 aaatccctgg taatccgttt tagaatccat gataataatt ttctggatta ttggtaattt
     6901 tttttgcacg ttcaaaattt tttgcaaccc ctttttggaa acaaacacta cggtaggctg
     6961 cgaaatgttc atactgttga gcaattcacg ttcattataa atgtcgttcg cgggcgcaac
     7021 tgcaactccg ataaataacg cgcccaacac cggcataaag aattgaagag a
//
