LOCUS       sequence-449342-        6764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of zebrafish Ago2 with D651E and Y680F 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 6764)
  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 6764)
  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-drAgo2DY-EF
FEATURES             Location/Qualifiers
     source          1..6764
                     /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             88..153
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    2816..2950
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2840..2859)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2894..2913
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3063..3083)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3063..3081)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3102..3118)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3102..3118)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3115..3137)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3126..3142
                     /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(3150..3156,3157..3174,3175..3180))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3195..3216
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3333..3350)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3504..4092)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3584..3603)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4263..5054,5055..5123))
                     /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     4886..4905
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5124..5228)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5254..5709)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5391..5412)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5603..5622
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5780..6764
                     /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 atccatggac tacaaagacc atgacggaga ttacaaagac
      121 cacgacatcg actacaagga tgacgatgac aagggaggat atcccattgg agcagctggt
      181 gctactgagc tgtttcaggg acgtccgtct tcagggtcag atgtttctgc tcctgcgtct
      241 cctccagcac cacaagaata tgtcttcaaa ccacctcagc ggccagactt tggcacaatg
      301 ggtaggacaa tcaagcttca ggccaacttc tttgaaatgg aaattcccaa gttggaggtt
      361 tatcattatg aaatagacat caagcctgag aaatgcccac ggagagttaa ccgtgaaata
      421 gttgagcaca tggtccagca ctttaaaacg cagatctttg gagatcgaaa gcctgtgtat
      481 gatgggagga agaatctcta cactgccatg cctttaccca ttggaagaga caaagtggag
      541 ctggaggtca cgattcctgg ggagggaaaa gacaggagct ttaaagtcgc tataaagtgg
      601 atgtcctgtg tcagtctgca agctttgcat gaagcactct ctggacggct accaaacatc
      661 cctttcgaga ccattcaggc tctggatgtt gtcatgagac atttgccctc tatgaggtac
      721 accccagtcg gacgttcatt cttcactcca tcggagggct gctctaaccc tcttggtgga
      781 ggaagagaag tttggtttgg ttttcatcag tctgttcggc catcactctg gaagatgatg
      841 ctcaacattg atgtgtctgc cactgcgttc tacaaagctc aacccgtcat tgaattcatg
      901 tgtgaggttt tggattttaa aagcatcgaa gagcagcaaa agcccttaac agattcccaa
      961 agagtgaagt ttaccaagga gatcaagggt ttgaaggttg aaattactca ctgtggacaa
     1021 atgaagagga agtacagagt gtgtaatgtg acgagaagac cagccagcca tcaaacgttt
     1081 cctttgcagc aagagaatgg tcagaccatt gaatgcactg tcgcccagta cttcaaggat
     1141 aagtacaaac tggtgcttcg atacccacat ctcccatgtt tacaagttgg tcaggagcag
     1201 aaacacacct accttccgtt ggaggtctgc aacattgtag ccgggcagcg atgcatcaag
     1261 aaactgacag acaatcagac ctccactatg atacgtgcca cagccaggtc tgcgcccgac
     1321 cgtcaggacg agatcagcaa actgatgaga agtgccaact tcaatacaga tccttatgtg
     1381 cgtgagtttg gagtcatggt aagagacgat atgactgaag tcaatggtcg ggttcttcaa
     1441 gcaccgtcga ttctctatgg tggaaggaac aaagcaatag caaccccagt ccagggcgtg
     1501 tgggacatga ggaacaagca gttccacaca gggattgaaa tcaaagtttg ggccattgcc
     1561 tgctttgccc cacagaggca gtgcacagaa ctcctgctga aggccttcac tgaccagctt
     1621 cgtaagatct cacgtgatgc tgggatgccc attcagggcc agccgtgctt ttgcaaatac
     1681 gcccagggag cagacagtgt ggagcccatg ttcaaacacc tcaagtacac ttatcaaggg
     1741 ttacagttgg tggtggtcat cttacccggc aagactcctg tttatgctga ggtgaagcgt
     1801 gttggagaca ctgttctcgg catggccacg cagtgtgtcc aggtgaagaa tgtgcagaag
     1861 accacacctc aaaccctttc taacctctgc ctcaagatta acgtcaaact gggtggagtc
     1921 aacaacattc ttcttccaca gggcaggccc ttggtgttcc agcaaccagt tatcttttta
     1981 ggtgcagatg tgactcatcc accagctgga gatggcaaga aaccctcaat tgctgctgtc
     2041 gttggcagta tggatgccca cccaagcaga tactgtgcca cagtgcgggt ccagcagcac
     2101 cggcaggaga tcattcagga tctggcaacc atggtcagag agttactcat ccagttctac
     2161 aaatccacac gcttcaaacc aacacgcatc atcttctaca gagacggcat ctctgaaggc
     2221 cagtttaacc aggttctaca gcatgagctg ctggctattc gtgaggcctg catcaagctg
     2281 gagaaagatt atcagccggg tatcaccttc gttgtagtac agaagagaca ccacaccaga
     2341 ctcttctgca tggacaggaa cgaacgggtt ggaaagagtg gaaacatccc tgctggcacc
     2401 actgtcgaca ccaaaattac tcatccattc gagtttgact tttatctttg cagtcatgct
     2461 ggaattcagg gaaccagtag gccgtcccac tatcatgtgc tgtgggacga caaccatttc
     2521 acctctgacg agctgcaggt cctcacctac cagctgtgcc acacctatgt tcgctgcacc
     2581 cgctcggtct ccattcctgc accagcctat tacgcccact tggtggcgtt tcgcgcccgc
     2641 taccacttgg ttgacaaaga acatgacagt gctgaaggca gtcacacatc aggtcagagt
     2701 aacggcagag atcaacaagc cctggccaaa gctgtgcaga tccaccagga cacactgcgc
     2761 accatgtact tcgcctgact cgagcctcta gaactatagt gagtcgtatt acgtagatcc
     2821 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     2881 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     2941 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     3001 ggaggttttt taattcgcgg ccgcggcgcc aatgcattgg gcccggtacc cagcttttgt
     3061 tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg
     3121 tgaaattgtt atccgctcac aattccacac aacatacgag ccgggagcat aaagtgtaaa
     3181 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     3241 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3301 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3361 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3421 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3481 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3541 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3601 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3661 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3721 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3781 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3841 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3901 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     3961 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4021 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4081 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4141 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4201 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4261 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4321 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4381 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     4441 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4501 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     4561 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     4621 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     4681 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     4741 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     4801 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     4861 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     4921 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     4981 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5041 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5101 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5161 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5221 gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa
     5281 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     5341 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     5401 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     5461 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     5521 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     5581 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     5641 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     5701 agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     5761 cctcttcgct attacgccag tcgaccgcca attcaatatg gcgtatatgg actcatgcca
     5821 attcaatatg gtggatctgg acctgtgcca attcaatatg gcgtatatgg actcgtgcca
     5881 attcaatatg gtggatctgg accccagcca attcaatatg gcggacttgg caccatgcca
     5941 attcaatatg gcggacctgg cactgtgcca actggggagg ggtctacttg gcacggtgcc
     6001 aagtttgagg aggggtcttg gccctgtgcc aagtccgcca tattgaattg gcatggtgcc
     6061 aataatggcg gccatattgg ctatatgcca ggatcaatat ataggcaata tccaatatgg
     6121 ccctatgcca atatggctat tggccaggtt caatactatg tattggccct atgccatata
     6181 gtattccata tatgggtttt cctattgacg tagatagccc ctcccaatgg gcggtcccat
     6241 ataccatata tggggcttcc taataccgcc catagccact cccccattga cgtcaatggt
     6301 ctctatatat ggtctttcct attgacgtca tatgggcggt cctattgacg tatatggcgc
     6361 ctcccccatt gacgtcaatt acggtaaatg gcccgcctgg ctcaatgccc attgacgtca
     6421 ataggaccac ccaccattga cgtcaatggg atggctcatt gcccattcat atccgttctc
     6481 acgcccccta ttgacgtcaa tgacggtaaa tggcccactt ggcagtacat caatatctat
     6541 taatagtaac ttggcaagta cattactatt ggaagtacgc cagggtacat tggcagtact
     6601 cccattgacg tcaatggcgg taaatggccc gcgatggctg ccaagtacat ccccattgac
     6661 gtcaatgggg aggggcaatg acgcaaatgg gcgttccatt gacgtaaatg ggcggtaggc
     6721 gtgcctaatg ggaggtctat ataagcaatg ctcgtttagg gaac
//
