LOCUS       sequence-449653-        6780 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human Ago2 with N-terminal 3xFLAG tag in zebrafish 
            embryos. Swapped N-domain for zebrafish Ago2 N-domain..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6780)
  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 6780)
  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-drN-hsAgo2
FEATURES             Location/Qualifiers
     source          1..6780
                     /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"
     regulatory      2125..2134
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2832..2966
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2856..2875)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2910..2929
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3079..3099)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3079..3097)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3118..3134)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3118..3134)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3131..3153)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3142..3158
                     /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(3166..3172,3173..3190,3191..3196))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3211..3232
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3349..3366)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3520..4108)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3600..3619)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4279..5070,5071..5139))
                     /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     4902..4921
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5140..5244)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5270..5725)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5407..5428)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5619..5638
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5796..6780
                     /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 actacaaaga tgacgatgac aagggaggat atcccattgg agcagctggt
      181 gctactgagc tgtttcaggg acgtccgtct tcagggtcag atgtctctgc 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 gtcatgaggc acttgccatc catgaggtac
      721 acccccgtgg gccgctcctt cttcaccgcg tccgaaggct gctctaaccc tcttggcggg
      781 ggccgagaag tgtggtttgg cttccatcag tccgtccggc cttctctctg gaaaatgatg
      841 ctgaatattg atgtgtcagc aacagcgttt tacaaggcac agccagtaat cgagtttgtt
      901 tgtgaagttt tggattttaa aagtattgaa gaacaacaaa aacctctgac agattcccaa
      961 agggtaaagt ttaccaaaga aattaaaggt ctaaaggtgg agataacgca ctgtgggcag
     1021 atgaagagga agtaccgcgt ctgcaatgtg acccggcggc ccgccagtca ccaaacattc
     1081 ccgctgcagc aggagagcgg gcagacggtg gagtgcacgg tggcccagta tttcaaggac
     1141 aggcacaagc ttgttctgcg ctacccccac ctcccatgtt tacaagtcgg acaggagcag
     1201 aaacacacct accttcccct ggaggtctgt aacattgtgg caggacaaag atgtattaaa
     1261 aaattaacgg acaatcagac ctcaaccatg atcagagcaa ctgctaggtc ggcgcccgat
     1321 cggcaagaag agattagcaa attgatgcga agtgcaagtt tcaacacaga tccatacgtc
     1381 cgtgaatttg gaatcatggt caaagatgag atgacagacg tgactgggcg ggtgctgcag
     1441 ccgccctcca tcctctacgg gggcaggaat aaagctattg cgacccctgt ccagggcgtc
     1501 tgggacatgc ggaacaagca gttccacacg ggcatcgaga tcaaggtgtg ggccattgcg
     1561 tgcttcgccc cccagcgcca gtgcacggaa gtccatctga agtccttcac agagcagctc
     1621 agaaagatct cgagagacgc tggcatgccc atccagggcc agccgtgctt ctgcaaatac
     1681 gcgcaggggg cggacagcgt ggagcccatg ttccggcacc tgaagaacac gtatgcgggc
     1741 ctgcagctgg tggtggtcat cctgcccggc aagacgcccg tgtacgccga ggtcaagcgc
     1801 gtgggagaca cggtgctggg gatggccacg cagtgcgtgc agatgaagaa cgtgcagagg
     1861 accacgccac agaccctgtc caacctctgc ctgaagatca acgtcaagct gggaggcgtg
     1921 aacaacatcc tgctgcccca gggcaggccg ccggtgttcc agcagcccgt catctttctg
     1981 ggagcagacg tcactcaccc ccccgccggg gatgggaaga agccctccat tgccgccgtg
     2041 gtgggcagca tggacgccca ccccaatcgc tactgcgcca ccgtgcgcgt gcagcagcac
     2101 cggcaggaga tcatacaaga cctggccgcc atggtccgcg agctcctcat ccagttctac
     2161 aagtccacgc gcttcaagcc cacccgcatc atcttctacc gcgacggtgt ctctgaaggc
     2221 cagttccagc aggttctcca ccacgagttg ctggccatcc gtgaggcctg tatcaagcta
     2281 gaaaaagact accagcccgg gatcaccttc atcgtggtgc agaagaggca ccacacccgg
     2341 ctcttctgca ctgacaagaa cgagcgggtt gggaaaagtg gaaacattcc agcaggcacg
     2401 actgtggaca cgaaaatcac ccaccccacc gagttcgact tctacctgtg tagtcacgct
     2461 ggcatccagg ggacaagcag gccttcgcac tatcacgtcc tctgggacga caatcgtttc
     2521 tcctctgatg agctgcagat cctaacctac cagctgtgtc acacctacgt gcgctgcaca
     2581 cgctccgtgt ccatcccagc gccagcatac tacgctcacc tggtggcctt ccgggccagg
     2641 taccacctgg tggataagga acatgacagt gctgaaggaa gccatacctc tgggcagagt
     2701 aacgggcgag accaccaagc actggccaag gcggtccagg ttcaccaaga cactctgcgc
     2761 accatgtact ttgcttgata ggatcgaatc ggactttcga attctagaac tatagtgagt
     2821 cgtattacgt agatccagac atgataagat acattgatga gtttggacaa accacaacta
     2881 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     2941 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     3001 ttcaggggga ggtgtgggag gttttttaat tcgcggccgc ggcgccaatg cattgggccc
     3061 ggtacccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc
     3121 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     3181 gagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     3241 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3301 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3361 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3421 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3481 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3541 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3601 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3661 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3721 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3781 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3841 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3901 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3961 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4021 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4081 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4141 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4201 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4261 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4321 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4381 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4441 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4501 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4561 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4621 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4681 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4741 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4801 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4861 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4921 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4981 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5041 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5101 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     5161 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5221 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     5281 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     5341 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     5401 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     5461 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     5521 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     5581 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     5641 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     5701 cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa
     5761 gggcgatcgg tgcgggcctc ttcgctatta cgccagtcga ccgccaattc aatatggcgt
     5821 atatggactc atgccaattc aatatggtgg atctggacct gtgccaattc aatatggcgt
     5881 atatggactc gtgccaattc aatatggtgg atctggaccc cagccaattc aatatggcgg
     5941 acttggcacc atgccaattc aatatggcgg acctggcact gtgccaactg gggaggggtc
     6001 tacttggcac ggtgccaagt ttgaggaggg gtcttggccc tgtgccaagt ccgccatatt
     6061 gaattggcat ggtgccaata atggcggcca tattggctat atgccaggat caatatatag
     6121 gcaatatcca atatggccct atgccaatat ggctattggc caggttcaat actatgtatt
     6181 ggccctatgc catatagtat tccatatatg ggttttccta ttgacgtaga tagcccctcc
     6241 caatgggcgg tcccatatac catatatggg gcttcctaat accgcccata gccactcccc
     6301 cattgacgtc aatggtctct atatatggtc tttcctattg acgtcatatg ggcggtccta
     6361 ttgacgtata tggcgcctcc cccattgacg tcaattacgg taaatggccc gcctggctca
     6421 atgcccattg acgtcaatag gaccacccac cattgacgtc aatgggatgg ctcattgccc
     6481 attcatatcc gttctcacgc cccctattga cgtcaatgac ggtaaatggc ccacttggca
     6541 gtacatcaat atctattaat agtaacttgg caagtacatt actattggaa gtacgccagg
     6601 gtacattggc agtactccca ttgacgtcaa tggcggtaaa tggcccgcga tggctgccaa
     6661 gtacatcccc attgacgtca atggggaggg gcaatgacgc aaatgggcgt tccattgacg
     6721 taaatgggcg gtaggcgtgc ctaatgggag gtctatataa gcaatgctcg tttagggaac
//
