LOCUS       sequence-449350-        6770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human Ago2 with E637D and F666Y mutations and 
            N-terminal 3xFLAG tag in zebrafish embryos..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6770)
  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 6770)
  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: pCS2+-FLAG3-hsAgo2EF-DY
FEATURES             Location/Qualifiers
     source          1..6770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             93..113
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             120..185
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      2115..2124
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2822..2956
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2846..2865)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2900..2919
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3069..3089)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3069..3087)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3108..3124)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3108..3124)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3121..3143)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3132..3148
                     /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(3156..3162,3163..3180,3181..3186))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3201..3222
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3339..3356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3510..4098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3590..3609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4269..5060,5061..5129))
                     /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     4892..4911
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5130..5234)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5260..5715)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5397..5418)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5609..5628
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5786..6770
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccaacga ccgaaaacct gtattttcag ggcgccatgg
      121 actacaaaga ccatgacgga gattacaaag accacgacat cgactacaag gatgacgatg
      181 acaagggagg atactcggga gccggccccg cacttgcacc tcctgcgccg ccgcccccca
      241 tccaaggata tgccttcaag cctccaccta gacccgactt tgggacctcc gggagaacaa
      301 tcaaattaca ggccaatttc ttcgaaatgg acatccccaa aattgacatc tatcattatg
      361 aattggatat caagccagag aagtgcccga ggagagttaa cagggaaatc gtggaacaca
      421 tggtccagca ctttaaaaca cagatctttg gggatcggaa gcccgtgttt gacggcagga
      481 agaatctata cacagccatg ccccttccga ttgggaggga caaggtggag ctggaggtca
      541 cgctgccagg agaaggcaag gatcgcatct tcaaggtgtc catcaagtgg gtgtcctgcg
      601 tgagcttgca ggcgttacac gatgcacttt cagggcggct gcctagcgtc ccttttgaga
      661 cgatccaggc cctggacgtg gtcatgaggc acttgccatc catgaggtac acccccgtgg
      721 gccgctcctt cttcaccgcg tccgaaggct gctctaaccc tcttggcggg ggccgagaag
      781 tgtggtttgg cttccatcag tccgtccggc cttctctctg gaaaatgatg ctgaatattg
      841 atgtgtcagc aacagcgttt tacaaggcac agccagtaat cgagtttgtt tgtgaagttt
      901 tggattttaa aagtattgaa gaacaacaaa aacctctgac agattcccaa agggtaaagt
      961 ttaccaaaga aattaaaggt ctaaaggtgg agataacgca ctgtgggcag atgaagagga
     1021 agtaccgcgt ctgcaatgtg acccggcggc ccgccagtca ccaaacattc ccgctgcagc
     1081 aggagagcgg gcagacggtg gagtgcacgg tggcccagta tttcaaggac aggcacaagc
     1141 ttgttctgcg ctacccccac ctcccatgtt tacaagtcgg acaggagcag aaacacacct
     1201 accttcccct ggaggtctgt aacattgtgg caggacaaag atgtattaaa aaattaacgg
     1261 acaatcagac ctcaaccatg atcagagcaa ctgctaggtc ggcgcccgat cggcaagaag
     1321 agattagcaa attgatgcga agtgcaagtt tcaacacaga tccatacgtc cgtgaatttg
     1381 gaatcatggt caaagatgag atgacagacg tgactgggcg ggtgctgcag ccgccctcca
     1441 tcctctacgg gggcaggaat aaagctattg cgacccctgt ccagggcgtc tgggacatgc
     1501 ggaacaagca gttccacacg ggcatcgaga tcaaggtgtg ggccattgcg tgcttcgccc
     1561 cccagcgcca gtgcacggaa gtccatctga agtccttcac agagcagctc agaaagatct
     1621 cgagagacgc tggcatgccc atccagggcc agccgtgctt ctgcaaatac gcgcaggggg
     1681 cggacagcgt ggagcccatg ttccggcacc tgaagaacac gtatgcgggc ctgcagctgg
     1741 tggtggtcat cctgcccggc aagacgcccg tgtacgccga ggtcaagcgc gtgggagaca
     1801 cggtgctggg gatggccacg cagtgcgtgc agatgaagaa cgtgcagagg accacgccac
     1861 agaccctgtc caacctctgc ctgaagatca acgtcaagct gggaggcgtg aacaacatcc
     1921 tgctgcccca gggcaggccg ccggtgttcc agcagcccgt catctttctg ggagcagacg
     1981 tcactcaccc ccccgccggg gatgggaaga agccctccat tgccgccgtg gtgggcagca
     2041 tggacgccca ccccaatcgc tactgcgcca ccgtgcgcgt gcagcagcac cggcaggata
     2101 tcatacaaga cctggccgcc atggtccgcg agctcctcat ccagttctac aagtccacgc
     2161 gcttcaagcc cacccgcatc atctattacc gcgacggtgt ctctgaaggc cagttccagc
     2221 aggttctcca ccacgagttg ctggccatcc gtgaggcctg tatcaagcta gaaaaagact
     2281 accagcccgg gatcaccttc atcgtggtgc agaagaggca ccacacccgg ctcttctgca
     2341 ctgacaagaa cgagcgggtt gggaaaagtg gaaacattcc agcaggcacg actgtggaca
     2401 cgaaaatcac ccaccccacc gagttcgact tctacctgtg tagtcacgct ggcatccagg
     2461 ggacaagcag gccttcgcac tatcacgtcc tctgggacga caatcgtttc tcctctgatg
     2521 agctgcagat cctaacctac cagctgtgtc acacctacgt gcgctgcaca cgctccgtgt
     2581 ccatcccagc gccagcatac tacgctcacc tggtggcctt ccgggccagg taccacctgg
     2641 tggataagga acatgacagt gctgaaggaa gccatacctc tgggcagagt aacgggcgag
     2701 accaccaagc actggccaag gcggtccagg ttcaccaaga cactctgcgc accatgtact
     2761 ttgcttgata ggatcgaatc ggactttcga attctagaac tatagtgagt cgtattacgt
     2821 agatccagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     2881 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     2941 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     3001 ggtgtgggag gttttttaat tcgcggccgc ggcgccaatg cattgggccc ggtacccagc
     3061 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     3121 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg gagcataaag
     3181 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3241 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3301 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3361 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3421 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3481 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3541 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3601 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3661 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     3721 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3781 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3841 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3901 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3961 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4021 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4081 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4141 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4201 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4261 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4321 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4381 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4441 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4501 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4561 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     4621 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     4681 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     4741 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     4801 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4861 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4921 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4981 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5041 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5101 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5161 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5221 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     5281 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     5341 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     5401 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     5461 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     5521 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     5581 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     5641 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     5701 cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     5761 tgcgggcctc ttcgctatta cgccagtcga ccgccaattc aatatggcgt atatggactc
     5821 atgccaattc aatatggtgg atctggacct gtgccaattc aatatggcgt atatggactc
     5881 gtgccaattc aatatggtgg atctggaccc cagccaattc aatatggcgg acttggcacc
     5941 atgccaattc aatatggcgg acctggcact gtgccaactg gggaggggtc tacttggcac
     6001 ggtgccaagt ttgaggaggg gtcttggccc tgtgccaagt ccgccatatt gaattggcat
     6061 ggtgccaata atggcggcca tattggctat atgccaggat caatatatag gcaatatcca
     6121 atatggccct atgccaatat ggctattggc caggttcaat actatgtatt ggccctatgc
     6181 catatagtat tccatatatg ggttttccta ttgacgtaga tagcccctcc caatgggcgg
     6241 tcccatatac catatatggg gcttcctaat accgcccata gccactcccc cattgacgtc
     6301 aatggtctct atatatggtc tttcctattg acgtcatatg ggcggtccta ttgacgtata
     6361 tggcgcctcc cccattgacg tcaattacgg taaatggccc gcctggctca atgcccattg
     6421 acgtcaatag gaccacccac cattgacgtc aatgggatgg ctcattgccc attcatatcc
     6481 gttctcacgc cccctattga cgtcaatgac ggtaaatggc ccacttggca gtacatcaat
     6541 atctattaat agtaacttgg caagtacatt actattggaa gtacgccagg gtacattggc
     6601 agtactccca ttgacgtcaa tggcggtaaa tggcccgcga tggctgccaa gtacatcccc
     6661 attgacgtca atggggaggg gcaatgacgc aaatgggcgt tccattgacg taaatgggcg
     6721 gtaggcgtgc ctaatgggag gtctatataa gcaatgctcg tttagggaac
//
