LOCUS       sequence-434402-        6599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter plasmid that encodes for the GFP fused to an N-terminal 
            flag-HA epitope (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: pAN008
FEATURES             Location/Qualifiers
     source          1..6599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    94..215
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(131..150)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     185..204
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(222..677)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(359..380)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     571..590
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        704..808
                     /gene="bla"
                     /label=AmpR promoter
     promoter        810..1167
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        810..1001
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(833..853)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1018..1153
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1202..1996
                     /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             1202..1996
                     /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(1256..1275)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1866..1885
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2184..2203)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2228..2275
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2604..3192
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3093..3112
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        3367..3670
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3671..3874
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3825..3845
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3957..3966
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3966..3989
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4005..4031
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4857..4910
                     /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             4917..6569
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /label=luciferase
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
     primer_bind     complement(4983..5001)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
ORIGIN
        1 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
       61 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
      121 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
      181 agttgtggtt tgtccaaact catcaatgta tcttaaggcg taaattgtaa gcgttaatat
      241 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
      301 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
      361 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
      421 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
      481 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
      541 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
      601 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
      661 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      721 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      781 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
      841 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
      901 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
      961 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     1021 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     1081 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     1141 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
     1201 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     1261 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     1321 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     1381 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     1441 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     1501 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     1561 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     1621 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     1681 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     1741 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     1801 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     1861 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     1921 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     1981 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     2041 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     2101 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     2161 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     2221 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
     2281 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     2341 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     2401 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
     2461 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     2521 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2581 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2641 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2701 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     2761 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2821 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2881 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2941 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3001 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3061 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3121 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3181 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3241 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3301 atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     3361 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     3421 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     3481 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     3541 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     3601 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     3661 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
     3721 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     3781 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     3841 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
     3901 gcgctaccgg actcagatct cgagctcaag cttcgaattc tgcagtcgac ggtaccgcca
     3961 ccatggacta caaagacgat gacgataaag ggggcggtgg aggttaccca tacgatgttc
     4021 cagattacgc tggtggaggt ggaggttctg cccccgccgc caagatcgag tgccgcatca
     4081 ccggcaccct gaacggcgtg gagttcgagc tggtgggcgg cggagagggc acccccgagc
     4141 agggccgcat gaccaacaag atgaagagca ccaaaggcgc cctgaccttc agcccctacc
     4201 tgctgagcca cgtgatgggc tacggcttct accacttcgg cacctacccc agcggctacg
     4261 agaacccctt cctgcacgcc atcaacaacg gcggctacac caacacccgc atcgagaagt
     4321 acgaggacgg cggcgtgctg cacgtgagct tcagctaccg ctacgaggcc ggccgcgtga
     4381 tcggcgactt caaggtggtg ggcaccggct tccccgagga cagcgtgatc ttcaccgaca
     4441 agatcatccg cagcaacgcc accgtggagc acctgcaccc catgggcgat aacgtgctgg
     4501 tgggcagctt cgcccgcacc ttcagcctgc gcgacggcgg ctactacagc ttcgtggtgg
     4561 acagccacat gcacttcaag agcgccatcc accccagcat cctgcagaac gggggcccca
     4621 tgttcgcctt ccgccgcgtg gaggagctgc acagcaacac cgagctgggc atcgtggagt
     4681 accagcacgc cttcaagacc cccatcgcct tcgccagatc tcgagatatc agccatggct
     4741 tcccgccggc ggtggcggcg caggatgatg gcacgctgcc catgtcttgt gcccaggaga
     4801 gcgggatgga ccgtcaccct gcagcctgtg cttctgctag gatcaatgtg accggtgagg
     4861 gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct tccggtatgg
     4921 aagacgccaa aaacataaag aaaggcccgg cgccattcta tccactagag gatggaaccg
     4981 ctggagagca actgcataag gctatgaaga gatacgccct ggttcctgga acaattgctt
     5041 ttacagatgc acatatcgag gtgaacatca cgtacgcgga atacttcgaa atgtccgttc
     5101 ggttggcaga agctatgaaa cgatatgggc tgaatacaaa tcacagaatc gtcgtatgca
     5161 gtgaaaactc tcttcaattc tttatgccgg tgttgggcgc gttatttatc ggagttgcag
     5221 ttgcgcccgc gaacgacatt tataatgaac gtgaattgct caacagtatg aacatttcgc
     5281 agcctaccgt agtgtttgtt tccaaaaagg ggttgcaaaa aattttgaac gtgcaaaaaa
     5341 aattaccaat aatccagaaa attattatca tggattctaa aacggattac cagggatttc
     5401 agtcgatgta cacgttcgtc acatctcatc tacctcccgg ttttaatgaa tacgattttg
     5461 taccagagtc ctttgatcgt gacaaaacaa ttgcactgat aatgaactcc tctggatcta
     5521 ctgggttacc taagggtgtg gcccttccgc atagaactgc ctgcgtcaga ttctcgcatg
     5581 ccagagatcc tatttttggc aatcaaatca ttccggatac tgcgatttta agtgttgttc
     5641 cattccatca cggttttgga atgtttacta cactcggata tttgatatgt ggatttcgag
     5701 tcgtcttaat gtatagattt gaagaagagc tgtttttacg atcccttcag gattacaaaa
     5761 ttcaaagtgc gttgctagta ccaaccctat tttcattctt cgccaaaagc actctgattg
     5821 acaaatacga tttatctaat ttacacgaaa ttgcttctgg gggcgcacct ctttcgaaag
     5881 aagtcgggga ggcggttgca aaacgcttcc atcttccagg gatacgacaa ggatatgggc
     5941 tcactgagac tacatcagct attctgatta cacccgaggg ggatgataaa ccgggcgcgg
     6001 tcggtaaagt tgttccattt tttgaagcga aggttgtgga tctggatacc gggaaaacgc
     6061 tgggcgttaa tcagagaggc gaattatgtg tcagaggacc tatgattatg tccggttatg
     6121 taaacaatcc ggaagcgacc aacgccttga ttgacaagga tggatggcta cattctggag
     6181 acatagctta ctgggacgaa gacgaacact tcttcatagt tgaccgcttg aagtctttaa
     6241 ttaaatacaa aggataccag gtggcccccg ctgaattgga gtcgatattg ttacaacacc
     6301 ccaacatctt cgacgcgggc gtggcaggtc ttcccgacga tgacgccggt gaacttcccg
     6361 ccgccgttgt tgttttggag cacggaaaga cgatgacgga aaaagagatc gtggattacg
     6421 tcgccagtca agtaacaacc gcgaaaaagt tgcgcggagg agttgtgttt gtggacgaag
     6481 taccgaaagg tcttaccgga aaactcgacg caagaaaaat cagagagatc ctcataaagg
     6541 ccaagaaggg cggaaagtcc aaattgtaat cgcggccgcg actctagatc ataatcagc
//
