LOCUS       sequence-434401-        7071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter plasmid that expresses a a firefly luciferase gene fused to
            exons 22-27 of the CFTR gene and a synthetic intron (positive 
            control). .
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: pAN056
FEATURES             Location/Qualifiers
     source          1..7071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     492..510
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     regulatory      573..582
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(665..868)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(694..714)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        869..1172
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(1347..1935)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1427..1446)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    2264..2311
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2336..2355
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(2543..3337)
                     /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(2543..3337)
                     /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(2654..2673)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3264..3283
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3372..3729)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3386..3521
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        3538..3729
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     3686..3706
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(3731..3835)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3862..4317
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3949..3968)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4159..4180
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4324..4445
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4335..4354)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4389..4408
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4572..4595)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(join(5998..7071,1..576))
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /label=luciferase
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
ORIGIN
        1 aattgttttg tcacgatcaa aggactctgg tacaaaatcg tattcattaa aaccgggagg
       61 tagatgagat gtgacgaacg tgtacatcga ctgaaatccc tggtaatccg ttttagaatc
      121 catgataata attttctgga ttattggtaa ttttttttgc acgttcaaaa ttttttgcaa
      181 cccctttttg gaaacaaaca ctacggtagg ctgcgaaatg ttcatactgt tgagcaattc
      241 acgttcatta taaatgtcgt tcgcgggcgc aactgcaact ccgataaata acgcgcccaa
      301 caccggcata aagaattgaa gagagttttc actgcatacg acgattctgt gatttgtatt
      361 cagcccatat cgtttcatag cttctgccaa ccgaacggac atttcgaagt attccgcgta
      421 cgtgatgttc acctcgatat gtgcatctgt aaaagcaatt gttccaggaa ccagggcgta
      481 tctcttcata gccttatgca gttgctctcc agcggttcca tcctctagtg gatagaatgg
      541 cgccgggcct ttctttatgt ttttggcgtc ttccatggtg gcggtaccgt cgactgcaga
      601 attcgaagct tgagctcgag atctgagtcc ggtagcgcta gcggatctga cggttcacta
      661 aaccagctct gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaatg
      721 gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc
      781 cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc
      841 cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt
      901 actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt
      961 taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca
     1021 agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg
     1081 ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc
     1141 caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta
     1201 atgaccccgt aattgattac tattaataac taatgcatgg cggtaatacg gttatccaca
     1261 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     1321 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     1381 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     1441 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     1501 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     1561 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     1621 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     1681 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     1741 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     1801 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     1861 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     1921 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     1981 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     2041 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta acctgaggct
     2101 atggcagggc ctgccgcccc gacgttggct gcgagccctg ggccttcacc cgaacttggg
     2161 gggtggggtg gggaaaagga agaaacgcgg gcgtattggc cccaatgggg tctcggtggg
     2221 gtatcgacag agtgccagcc ctgggaccga accccgcgtt tatgaacaaa cgacccaaca
     2281 ccgtgcgttt tattctgtct ttttattgcc gtcatagcgc gggttccttc cggtattgtc
     2341 tccttccgtg tttcagttag cctcccccta gggtgggcga agaactccag catgagatcc
     2401 ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca
     2461 tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc
     2521 atttcgaacc ccagagtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc
     2581 gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc
     2641 caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac
     2701 ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca
     2761 agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgctc gccttgagcc
     2821 tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga
     2881 caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga
     2941 atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata
     3001 ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata
     3061 gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg
     3121 tcgtggccag ccacgatagc cgcgctgcct cgtcttgcag ttcattcagg gcaccggaca
     3181 ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat
     3241 cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg
     3301 ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct catcctgtct
     3361 cttgatcgat ctttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat
     3421 agctcagagg ccgaggcggc ctcggcctct gcataaataa aaaaaattag tcagccatgg
     3481 ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt taggggcggg
     3541 actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct
     3601 ggggactttc cacacctggt tgctgactaa ttgagatgca tgctttgcat acttctgcct
     3661 gctggggagc ctggggactt tccacaccct aactgacaca cattccacag ctggttcttt
     3721 ccgcctcagg actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     3781 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     3841 ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3901 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3961 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4021 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     4081 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4141 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     4201 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     4261 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttacg
     4321 ccttaagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     4381 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     4441 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     4501 ttttttaaag caagtaaaac ctctacaaat gtggtatggc tgattatgat ctagagtcgc
     4561 ggccgcgatt atttatcgtc atcgtctttg tagtcgctac cgcccccaag ccttgtatct
     4621 tgcacctctt cttctgtctc ctctttcaga gcagcaatct ggggcttaga cttgcacttg
     4681 cttgagttcc ggtggggaaa gagcttcacc ctgtcggagg ggctgatggc ttgccggaag
     4741 aggctcctct cgttcagcag tttctggatg gaatcgtact gccgcacttt gttctcttct
     4801 atgaccaaaa attgttggca ttccagcatt gcttctatcc tgtgttcaca gagaattact
     4861 gtgcaatcag caaatgcttg ttttagagtt cttctaatta tttggtatgt tactggatcc
     4921 aaatgagcac tgggttcatc aagcagcaag atcttcgcct tactgagaac agatctagcc
     4981 aagcacatca actgcttgtg gccatggctt aggacacagc ccccatccac aaggacaaag
     5041 tcaagcttcc caggaaactg ttctatcaca gatctgagcc caacctgcag ttagagaaga
     5101 tagttcagat accacagagt taaaaacact caagcagttg acattctaaa aataaacaca
     5161 ggagttctga aagcttatgt ttttcatacc aatccctcta cccccactac ctgccaccat
     5221 ttgaattgat tgagcaaaag tctatttaca accttgacag ctcaaaagaa tcagtctata
     5281 acttgttacc tctttcaaaa tcatttcagt taggggtagg tccagtcaaa agtacctgtt
     5341 gctccaggta tgttagggta ctccatttta aagttgtgca cacacataca tgcacacaaa
     5401 gtgtgtagaa tgatgtcagc tatatcagcc atttgtgttg gtatgagtta cccctttcaa
     5461 aatcatttca gttagcagcc ttacctcatc tgcaactttc catatttctt gatcactcca
     5521 ctgttcatag ggatccaagt tttttctaaa tgttccagaa aaaataaata ctttctgtgg
     5581 tatcactcca aaggctttcc tccactgttg caaagttatt gaatcccaag acacaccatc
     5641 gatctggatt tctccttcag tgttcagtag tctcaaaaaa gctgataaca aagtactctt
     5701 ccctgatcca gttcttccca agaggcccac cctctggcca ggacttattg agaaggaaat
     5761 gttctctaat atggcatttc caccttctgt gtattttgct gtgagatctt tgacagtcat
     5821 ttggccccct gagggccaga tgtcatcttt cttcacgtgt gaattctcaa taatcataac
     5881 tttcgagagt tggccattct tgtatggttt ggttgacttg gtaggtttac cttctgttgg
     5941 catgtcaatg aacttaaaga ctcggctcac agatcgcatg ctacctccac tgcctcccaa
     6001 tttggacttt ccgcccttct tggcctttat gaggatctct ctgatttttc ttgcgtcgag
     6061 ttttccggta agacctttcg gtacttcgtc cacaaacaca actcctccgc gcaacttttt
     6121 cgcggttgtt acttgactgg cgacgtaatc cacgatctct ttttccgtca tcgtctttcc
     6181 gtgctccaaa acaacaacgg cggcgggaag ttcaccggcg tcatcgtcgg gaagacctgc
     6241 cacgcccgcg tcgaagatgt tggggtgttg taacaatatc gactccaatt cagcgggggc
     6301 cacctggtat cctttgtatt taattaaaga cttcaagcgg tcaactatga agaagtgttc
     6361 gtcttcgtcc cagtaagcta tgtctccaga atgtagccat ccatccttgt caatcaaggc
     6421 gttggtcgct tccggattgt ttacataacc ggacataatc ataggtcctc tgacacataa
     6481 ttcgcctctc tgattaacgc ccagcgtttt cccggtatcc agatccacaa ccttcgcttc
     6541 aaaaaatgga acaactttac cgaccgcgcc cggtttatca tccccctcgg gtgtaatcag
     6601 aatagctgat gtagtctcag tgagcccata tccttgtcgt atccctggaa gatggaagcg
     6661 ttttgcaacc gcctccccga cttctttcga aagaggtgcg cccccagaag caatttcgtg
     6721 taaattagat aaatcgtatt tgtcaatcag agtgcttttg gcgaagaatg aaaatagggt
     6781 tggtactagc aacgcacttt gaattttgta atcctgaagg gatcgtaaaa acagctcttc
     6841 ttcaaatcta tacattaaga cgactcgaaa tccacatatc aaatatccga gtgtagtaaa
     6901 cattccaaaa ccgtgatgga atggaacaac acttaaaatc gcagtatccg gaatgatttg
     6961 attgccaaaa ataggatctc tggcatgcga gaatctgacg caggcagttc tatgcggaag
     7021 ggccacaccc ttaggtaacc cagtagatcc agaggagttc attatcagtg c
//
