LOCUS       sequence-449651-        6754 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of zebrafish Ago2 with N-terminal 3xFLAG tag in zebrafish
            embryos. Swapped N-domain for human Ago2 N-domain..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6754)
  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 6754)
  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-hsN-drAgo2
FEATURES             Location/Qualifiers
     source          1..6754
                     /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"
     polyA_signal    2806..2940
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2830..2849)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2884..2903
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3053..3073)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3053..3071)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3092..3108)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3092..3108)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3105..3127)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3116..3132
                     /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(3140..3146,3147..3164,3165..3170))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3185..3206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3323..3340)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3494..4082)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3574..3593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4253..5044,5045..5113))
                     /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     4876..4895
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5114..5218)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5244..5699)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5381..5402)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5593..5612
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5770..6754
                     /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 gtcatgagac atttgccctc tatgaggtac accccagtcg
      721 gacgttcatt cttcactcca tcggagggct gctctaaccc tcttggtgga ggaagagaag
      781 tttggtttgg ttttcatcag tctgttcggc catcactctg gaagatgatg ctcaacattg
      841 atgtgtctgc cactgcgttc tacaaagctc aacccgtcat tgaattcatg tgtgaggttt
      901 tggattttaa aagcatcgaa gagcagcaaa agcccttaac agattcccaa agagtgaagt
      961 ttaccaagga gatcaagggt ttgaaggttg aaattactca ctgtggacaa atgaagagga
     1021 agtacagagt gtgtaatgtg acgagaagac cagccagcca tcaaacgttt cctttgcagc
     1081 aagagaatgg tcagaccatt gaatgcactg tcgcccagta cttcaaggat aagtacaaac
     1141 tggtgcttcg atacccacat ctcccatgtt tacaagttgg tcaggagcag aaacacacct
     1201 accttccgtt ggaggtctgc aacattgtag ccgggcagcg atgcatcaag aaactgacag
     1261 acaatcagac ctccactatg atacgtgcca cagccaggtc tgcgcccgac cgtcaggacg
     1321 agatcagcaa actgatgaga agtgccaact tcaatacaga tccttatgtg cgtgagtttg
     1381 gagtcatggt aagagacgat atgactgaag tcaatggtcg ggttcttcaa gcaccgtcga
     1441 ttctctatgg tggaaggaac aaagcaatag caaccccagt ccagggcgtg tgggacatga
     1501 ggaacaagca gttccacaca gggattgaaa tcaaagtttg ggccattgcc tgctttgccc
     1561 cacagaggca gtgcacagaa ctcctgctga aggccttcac tgaccagctt cgtaagatct
     1621 cacgtgatgc tgggatgccc attcagggcc agccgtgctt ttgcaaatac gcccagggag
     1681 cagacagtgt ggagcccatg ttcaaacacc tcaagtacac ttatcaaggg ttacagttgg
     1741 tggtggtcat cttacccggc aagactcctg tttatgctga ggtgaagcgt gttggagaca
     1801 ctgttctcgg catggccacg cagtgtgtcc aggtgaagaa tgtgcagaag accacacctc
     1861 aaaccctttc taacctctgc ctcaagatta acgtcaaact gggtggagtc aacaacattc
     1921 ttcttccaca gggcaggccc ttggtgttcc agcaaccagt tatcttttta ggtgcagatg
     1981 tgactcatcc accagctgga gatggcaaga aaccctcaat tgctgctgtc gttggcagta
     2041 tggatgccca cccaagcaga tactgtgcca cagtgcgggt ccagcagcac cggcaggaca
     2101 tcattcagga tctggcaacc atggtcagag agttactcat ccagttctac aaatccacac
     2161 gcttcaaacc aacacgcatc atctactaca gagacggcat ctctgaaggc cagtttaacc
     2221 aggttctaca gcatgagctg ctggctattc gtgaggcctg catcaagctg gagaaagatt
     2281 atcagccggg tatcaccttc gttgtagtac agaagagaca ccacaccaga ctcttctgca
     2341 tggacaggaa cgaacgggtt ggaaagagtg gaaacatccc tgctggcacc actgtcgaca
     2401 ccaaaattac tcatccattc gagtttgact tttatctttg cagtcatgct ggaattcagg
     2461 gaaccagtag gccgtcccac tatcatgtgc tgtgggacga caaccatttc acctctgacg
     2521 agctgcaggt cctcacctac cagctgtgcc acacctatgt tcgctgcacc cgctcggtct
     2581 ccattcctgc accagcctat tacgcccact tggtggcgtt tcgcgcccgc taccacttgg
     2641 ttgacaaaga acatgacagt gctgaaggca gtcacacatc aggtcagagt aacggcagag
     2701 atcaacaagc cctggccaaa gctgtgcaga tccaccagga cacactgcgc accatgtact
     2761 tcgcctgact cgagcctcta gaactatagt gagtcgtatt acgtagatcc agacatgata
     2821 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     2881 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     2941 aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
     3001 taattcgcgg ccgcggcgcc aatgcattgg gcccggtacc cagcttttgt tccctttagt
     3061 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3121 atccgctcac aattccacac aacatacgag ccgggagcat aaagtgtaaa gcctggggtg
     3181 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3241 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3301 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3361 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3421 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3481 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3541 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3601 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3661 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3721 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3781 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3841 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3901 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     3961 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4021 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4081 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4141 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4201 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4261 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4321 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4381 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4441 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4501 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4561 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4621 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4681 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4741 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4801 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4861 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4921 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4981 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5041 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5101 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5161 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5221 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     5281 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     5341 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     5401 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     5461 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     5521 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     5581 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     5641 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     5701 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     5761 attacgccag tcgaccgcca attcaatatg gcgtatatgg actcatgcca attcaatatg
     5821 gtggatctgg acctgtgcca attcaatatg gcgtatatgg actcgtgcca attcaatatg
     5881 gtggatctgg accccagcca attcaatatg gcggacttgg caccatgcca attcaatatg
     5941 gcggacctgg cactgtgcca actggggagg ggtctacttg gcacggtgcc aagtttgagg
     6001 aggggtcttg gccctgtgcc aagtccgcca tattgaattg gcatggtgcc aataatggcg
     6061 gccatattgg ctatatgcca ggatcaatat ataggcaata tccaatatgg ccctatgcca
     6121 atatggctat tggccaggtt caatactatg tattggccct atgccatata gtattccata
     6181 tatgggtttt cctattgacg tagatagccc ctcccaatgg gcggtcccat ataccatata
     6241 tggggcttcc taataccgcc catagccact cccccattga cgtcaatggt ctctatatat
     6301 ggtctttcct attgacgtca tatgggcggt cctattgacg tatatggcgc ctcccccatt
     6361 gacgtcaatt acggtaaatg gcccgcctgg ctcaatgccc attgacgtca ataggaccac
     6421 ccaccattga cgtcaatggg atggctcatt gcccattcat atccgttctc acgcccccta
     6481 ttgacgtcaa tgacggtaaa tggcccactt ggcagtacat caatatctat taatagtaac
     6541 ttggcaagta cattactatt ggaagtacgc cagggtacat tggcagtact cccattgacg
     6601 tcaatggcgg taaatggccc gcgatggctg ccaagtacat ccccattgac gtcaatgggg
     6661 aggggcaatg acgcaaatgg gcgttccatt gacgtaaatg ggcggtaggc gtgcctaatg
     6721 ggaggtctat ataagcaatg ctcgtttagg gaac
//
