LOCUS       sequence-434399-        6599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter plasmid that expresses GFP and firefly luciferase linked 
            with a viral 2A peptide (positive control). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6599)
  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 6599)
  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: pAN012
FEATURES             Location/Qualifiers
     source          1..6599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        109..412
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(587..1175)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(667..686)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    1504..1551
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     1576..1595
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(1783..2577)
                     /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(1783..2577)
                     /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(1894..1913)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2504..2523
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2612..2969)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2626..2761
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        2778..2969
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     2926..2946
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(2971..3075)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3102..3557
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3189..3208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3399..3420
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    3564..3685
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3575..3594)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3629..3648
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3809..5461)
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /label=luciferase
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
     primer_bind     5377..5395
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     CDS             complement(5468..5521)
                     /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(5663..6328)
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     CDS             complement(5663..6328)
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     CDS             complement(6347..6373)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(6389..6412)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6412..6421
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(join(6504..6599,1..108))
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6533..6553)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 ttggtgccaa aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga
       61 gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc
      121 gatgactaat acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc
      181 ataatgccag gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg
      241 atacacttga tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca
      301 atggaaagtc cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg
      361 ggggtcgttg ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc
      421 catatatggg ctatgaacta atgaccccgt aattgattac tattaataac taatgcatgg
      481 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
      541 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
      601 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
      661 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
      721 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
      781 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
      841 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
      901 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
      961 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     1021 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     1081 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     1141 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     1201 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     1261 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     1321 tatatgagta acctgaggct atggcagggc ctgccgcccc gacgttggct gcgagccctg
     1381 ggccttcacc cgaacttggg gggtggggtg gggaaaagga agaaacgcgg gcgtattggc
     1441 cccaatgggg tctcggtggg gtatcgacag agtgccagcc ctgggaccga accccgcgtt
     1501 tatgaacaaa cgacccaaca ccgtgcgttt tattctgtct ttttattgcc gtcatagcgc
     1561 gggttccttc cggtattgtc tccttccgtg tttcagttag cctcccccta gggtgggcga
     1621 agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga
     1681 ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt
     1741 tgggcgtcgc ttggtcggtc atttcgaacc ccagagtccc gctcagaaga actcgtcaag
     1801 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa
     1861 gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc
     1921 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt
     1981 ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc
     2041 gggcatgctc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc
     2101 gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg
     2161 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat
     2221 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg
     2281 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac
     2341 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcttgcag
     2401 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga
     2461 cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
     2521 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg
     2581 aaacgatcct catcctgtct cttgatcgat ctttgcaaaa gcctaggcct ccaaaaaagc
     2641 ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa
     2701 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
     2761 tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac
     2821 ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca
     2881 tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca
     2941 cattccacag ctggttcttt ccgcctcagg actcttcctt tttcaatatt attgaagcat
     3001 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     3061 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc
     3121 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3181 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3241 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3301 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3361 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3421 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     3481 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     3541 attaacgctt acaatttacg ccttaagata cattgatgag tttggacaaa ccacaactag
     3601 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     3661 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     3721 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc
     3781 tgattatgat ctagagtcgc ggccgcgatt acaatttgga ctttccgccc ttcttggcct
     3841 ttatgaggat ctctctgatt tttcttgcgt cgagttttcc ggtaagacct ttcggtactt
     3901 cgtccacaaa cacaactcct ccgcgcaact ttttcgcggt tgttacttga ctggcgacgt
     3961 aatccacgat ctctttttcc gtcatcgtct ttccgtgctc caaaacaaca acggcggcgg
     4021 gaagttcacc ggcgtcatcg tcgggaagac ctgccacgcc cgcgtcgaag atgttggggt
     4081 gttgtaacaa tatcgactcc aattcagcgg gggccacctg gtatcctttg tatttaatta
     4141 aagacttcaa gcggtcaact atgaagaagt gttcgtcttc gtcccagtaa gctatgtctc
     4201 cagaatgtag ccatccatcc ttgtcaatca aggcgttggt cgcttccgga ttgtttacat
     4261 aaccggacat aatcataggt cctctgacac ataattcgcc tctctgatta acgcccagcg
     4321 ttttcccggt atccagatcc acaaccttcg cttcaaaaaa tggaacaact ttaccgaccg
     4381 cgcccggttt atcatccccc tcgggtgtaa tcagaatagc tgatgtagtc tcagtgagcc
     4441 catatccttg tcgtatccct ggaagatgga agcgttttgc aaccgcctcc ccgacttctt
     4501 tcgaaagagg tgcgccccca gaagcaattt cgtgtaaatt agataaatcg tatttgtcaa
     4561 tcagagtgct tttggcgaag aatgaaaata gggttggtac tagcaacgca ctttgaattt
     4621 tgtaatcctg aagggatcgt aaaaacagct cttcttcaaa tctatacatt aagacgactc
     4681 gaaatccaca tatcaaatat ccgagtgtag taaacattcc aaaaccgtga tggaatggaa
     4741 caacacttaa aatcgcagta tccggaatga tttgattgcc aaaaatagga tctctggcat
     4801 gcgagaatct gacgcaggca gttctatgcg gaagggccac acccttaggt aacccagtag
     4861 atccagagga gttcattatc agtgcaattg ttttgtcacg atcaaaggac tctggtacaa
     4921 aatcgtattc attaaaaccg ggaggtagat gagatgtgac gaacgtgtac atcgactgaa
     4981 atccctggta atccgtttta gaatccatga taataatttt ctggattatt ggtaattttt
     5041 tttgcacgtt caaaattttt tgcaacccct ttttggaaac aaacactacg gtaggctgcg
     5101 aaatgttcat actgttgagc aattcacgtt cattataaat gtcgttcgcg ggcgcaactg
     5161 caactccgat aaataacgcg cccaacaccg gcataaagaa ttgaagagag ttttcactgc
     5221 atacgacgat tctgtgattt gtattcagcc catatcgttt catagcttct gccaaccgaa
     5281 cggacatttc gaagtattcc gcgtacgtga tgttcacctc gatatgtgca tctgtaaaag
     5341 caattgttcc aggaaccagg gcgtatctct tcatagcctt atgcagttgc tctccagcgg
     5401 ttccatcctc tagtggatag aatggcgccg ggcctttctt tatgtttttg gcgtcttcca
     5461 taccggaagg gccgggattc tcctccacgt caccgcatgt tagaagactt cctctgccct
     5521 caccggtcac attgatccta gcagaagcac aggctgcagg gtgacggtcc atcccgctct
     5581 cctgggcaca agacatgggc agcgtgccat catcctgcgc cgccaccgcc ggcgggaagc
     5641 catggctgat atctcgagat ctggcgaagg cgatgggggt cttgaaggcg tgctggtact
     5701 ccacgatgcc cagctcggtg ttgctgtgca gctcctccac gcggcggaag gcgaacatgg
     5761 ggcccccgtt ctgcaggatg ctggggtgga tggcgctctt gaagtgcatg tggctgtcca
     5821 ccacgaagct gtagtagccg ccgtcgcgca ggctgaaggt gcgggcgaag ctgcccacca
     5881 gcacgttatc gcccatgggg tgcaggtgct ccacggtggc gttgctgcgg atgatcttgt
     5941 cggtgaagat cacgctgtcc tcggggaagc cggtgcccac caccttgaag tcgccgatca
     6001 cgcggccggc ctcgtagcgg tagctgaagc tcacgtgcag cacgccgccg tcctcgtact
     6061 tctcgatgcg ggtgttggtg tagccgccgt tgttgatggc gtgcaggaag gggttctcgt
     6121 agccgctggg gtaggtgccg aagtggtaga agccgtagcc catcacgtgg ctcagcaggt
     6181 aggggctgaa ggtcagggcg cctttggtgc tcttcatctt gttggtcatg cggccctgct
     6241 cgggggtgcc ctctccgccg cccaccagct cgaactccac gccgttcagg gtgccggtga
     6301 tgcggcactc gatcttcatg gcgggcatag aacctccacc tccaccagcg taatctggaa
     6361 catcgtatgg gtaacctcca ccgccccctt tatcgtcatc gtctttgtag tccatggtgg
     6421 cggtaccgtc gactgcagaa ttcgaagctt gagctcgaga tctgagtccg gtagcgctag
     6481 cggatctgac ggttcactaa accagctctg cttatataga cctcccaccg tacacgccta
     6541 ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt ttggaaagtc ccgttgatt
//
