LOCUS       sequence-449354-        8113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of zebrafish Ago2 with D651E and Y680F mutations and 
            N-terminal 3xFLAG tag in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8113)
  AUTHORS   Chen GR, Sive H, Bartel DP
  TITLE     A Seed Mismatch Enhances Argonaute2-Catalyzed Cleavage and Partially
            Rescues Severely Impaired Cleavage Found in Fish.
  JOURNAL   Mol Cell. 2017 Dec 21;68(6):1095-1107.e5. doi: 
            10.1016/j.molcel.2017.11.032.
  PUBMED    29272705
REFERENCE   2  (bases 1 to 8113)
  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"; doi: 
            "10.1016/j.molcel.2017.11"; journalName: "Mol Cell"; date: 
            "2017-12-21- 21"; volume: "68"; issue: "6"; pages: "1095-1107.e5"
COMMENT     Sequence Label: pcDNA3-FLAG3-drAgo2DY-EF
FEATURES             Location/Qualifiers
     source          1..8113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     815..839
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      927..936
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             936..1001
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3684..3703)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3728..3776
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3978..4406
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4065..4084)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4275..4296
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4415..4435)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4420..4749
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4600..4735
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4662..4681
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4816..5610
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4870..4889)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5480..5499
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5786..5907
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5823..5842)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5877..5896
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5956..5972)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5956..5972)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5969..5991)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5980..5996
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6004..6010,6011..6028,6029..6034))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6049..6070
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6187..6204)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6358..6946)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6438..6457)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7117..7908,7909..7977))
                     /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     7740..7759
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7978..8082)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg
      841 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      901 tccggactct agaggatcga acccttgcca ccatggacta caaagaccat gacggtgatt
      961 ataaagatca tgatatcgat tacaaggatg acgatgacaa gcttggtggt tcttatccca
     1021 ttggagcagc tggtgctact gagctgtttc agggacgtcc gtcttcaggg tcagatgttt
     1081 ctgctcctgc gtctcctcca gcaccacaag aatatgtctt caaaccacct cagcggccag
     1141 actttggcac aatgggtagg acaatcaagc ttcaggccaa cttctttgaa atggaaattc
     1201 ccaagttgga ggtttatcat tatgaaatag acatcaagcc tgagaaatgc ccacggagag
     1261 ttaaccgtga aatagttgag cacatggtcc agcactttaa aacgcagatc tttggagatc
     1321 gaaagcctgt gtatgatggg aggaagaatc tctacactgc catgccttta cccattggaa
     1381 gagacaaagt ggagctggag gtcacgattc ctggggaggg aaaagacagg agctttaaag
     1441 tcgctataaa gtggatgtcc tgtgtcagtc tgcaagcttt gcatgaagca ctctctggac
     1501 ggctaccaaa catccctttc gagaccattc aggctctgga tgttgtcatg agacatttgc
     1561 cctctatgag gtacacccca gtcggacgtt cattcttcac tccatcggag ggctgctcta
     1621 accctcttgg tggaggaaga gaagtttggt ttggttttca tcagtctgtt cggccatcac
     1681 tctggaagat gatgctcaac attgatgtgt ctgccactgc gttctacaaa gctcaacccg
     1741 tcattgaatt catgtgtgag gttttggatt ttaaaagcat cgaagagcag caaaagccct
     1801 taacagattc ccaaagagtg aagtttacca aggagatcaa gggtttgaag gttgaaatta
     1861 ctcactgtgg acaaatgaag aggaagtaca gagtgtgtaa tgtgacgaga agaccagcca
     1921 gccatcaaac gtttcctttg cagcaagaga atggtcagac cattgaatgc actgtcgccc
     1981 agtacttcaa ggataagtac aaactggtgc ttcgataccc acatctccca tgtttacaag
     2041 ttggtcagga gcagaaacac acctaccttc cgttggaggt ctgcaacatt gtagccgggc
     2101 agcgatgcat caagaaactg acagacaatc agacctccac tatgatacgt gccacagcca
     2161 ggtctgcgcc cgaccgtcag gacgagatca gcaaactgat gagaagtgcc aacttcaata
     2221 cagatcctta tgtgcgtgag tttggagtca tggtaagaga cgatatgact gaagtcaatg
     2281 gtcgggttct tcaagcaccg tcgattctct atggtggaag gaacaaagca atagcaaccc
     2341 cagtccaggg cgtgtgggac atgaggaaca agcagttcca cacagggatt gaaatcaaag
     2401 tttgggccat tgcctgcttt gccccacaga ggcagtgcac agaactcctg ctgaaggcct
     2461 tcactgacca gcttcgtaag atctcacgtg atgctgggat gcccattcag ggccagccgt
     2521 gcttttgcaa atacgcccag ggagcagaca gtgtggagcc catgttcaaa cacctcaagt
     2581 acacttatca agggttacag ttggtggtgg tcatcttacc cggcaagact cctgtttatg
     2641 ctgaggtgaa gcgtgttgga gacactgttc tcggcatggc cacgcagtgt gtccaggtga
     2701 agaatgtgca gaagaccaca cctcaaaccc tttctaacct ctgcctcaag attaacgtca
     2761 aactgggtgg agtcaacaac attcttcttc cacagggcag gcccttggtg ttccagcaac
     2821 cagttatctt tttaggtgca gatgtgactc atccaccagc tggagatggc aagaaaccct
     2881 caattgctgc tgtcgttggc agtatggatg cccacccaag cagatactgt gccacagtgc
     2941 gggtccagca gcaccggcag gagatcattc aggatctggc aaccatggtc agagagttac
     3001 tcatccagtt ctacaaatcc acacgcttca aaccaacacg catcatcttc tacagagacg
     3061 gcatctctga aggccagttt aaccaggttc tacagcatga gctgctggct attcgtgagg
     3121 cctgcatcaa gctggagaaa gattatcagc cgggtatcac cttcgttgta gtacagaaga
     3181 gacaccacac cagactcttc tgcatggaca ggaacgaacg ggttggaaag agtggaaaca
     3241 tccctgctgg caccactgtc gacaccaaaa ttactcatcc attcgagttt gacttttatc
     3301 tttgcagtca tgctggaatt cagggaacca gtaggccgtc ccactatcat gtgctgtggg
     3361 acgacaacca tttcacctct gacgagctgc aggtcctcac ctaccagctg tgccacacct
     3421 atgttcgctg cacccgctcg gtctccattc ctgcaccagc ctattacgcc cacttggtgg
     3481 cgtttcgcgc ccgctaccac ttggttgaca aagaacatga cagtgctgaa ggcagtcaca
     3541 catcaggtca gagtaacggc agagatcaac aagccctggc caaagctgtg cagatccacc
     3601 aggacacact gcgcaccatg tacttcgcct gaaagggttc gatccctacc ggttagtaat
     3661 gagtttaaac gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     3721 gctatgacgg caataaaaag acagaataaa acgcacgggt gttgggtcgt ttgttcataa
     3781 acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg
     3841 ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc
     3901 agggctcgca gccaacgtcg gggcggcagg ccctgccata gcagatctgc gcagctgggg
     3961 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4021 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     4081 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     4141 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     4201 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4261 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     4321 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     4381 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     4441 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4501 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4561 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     4621 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4681 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     4741 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     4801 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     4861 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     4921 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     4981 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     5041 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     5101 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     5161 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     5221 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     5281 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     5341 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     5401 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     5461 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     5521 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     5581 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgcg aaatgaccga
     5641 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     5701 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     5761 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     5821 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     5881 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     5941 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     6001 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     6061 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6121 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     6181 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     6241 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     6301 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     6361 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     6421 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6481 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6541 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6601 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6661 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6721 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6781 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6841 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6901 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6961 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     7021 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     7081 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     7141 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     7201 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     7261 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     7321 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     7381 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     7441 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     7501 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7561 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7621 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7681 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     7741 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7801 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7861 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7921 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7981 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     8041 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     8101 gccacctgac gtc
//
