LOCUS       sequence-449652-        6855 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of zebrafish Ago2 with D683A mutation and N-terminal 
            3xFLAG tag in zebrafish embryos..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6855)
  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 6855)
  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-drAgo2D-A
FEATURES             Location/Qualifiers
     source          1..6855
                     /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    2907..3041
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2931..2950)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2985..3004
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3154..3174)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3154..3172)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3193..3209)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3193..3209)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3206..3228)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3217..3233
                     /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(3241..3247,3248..3265,3266..3271))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3286..3307
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3424..3441)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3595..4183)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3675..3694)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4354..5145,5146..5214))
                     /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     4977..4996
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5215..5319)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5345..5800)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5482..5503)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5694..5713
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5871..6855
                     /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 cggcaggaca tcattcagga tctggcaacc atggtcagag agttactcat ccagttctac
     2161 aaatccacac gcttcaaacc aacacgcatc atctactaca gagccggcat 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 cgactcgaga atctagacta cctgcactgt gagcactttg
     2821 ataagactac ctgcactgtg agcactttga taagactacc tgcactgtga gcactttgct
     2881 agaactatag tgagtcgtat tacgtagatc cagacatgat aagatacatt gatgagtttg
     2941 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     3001 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     3061 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaattcgcg gccgcggcgc
     3121 caatgcattg ggcccggtac ccagcttttg ttccctttag tgagggttaa ttgcgcgctt
     3181 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3241 caacatacga gccgggagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3301 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3361 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3421 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3481 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3541 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3601 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3661 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3721 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3781 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3841 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3901 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3961 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4021 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4081 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4141 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4201 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4261 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4321 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4381 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4441 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4501 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4561 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4621 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4681 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4741 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     4801 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4861 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4921 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4981 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5041 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5101 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5161 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5221 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5281 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5341 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5401 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5461 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     5521 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     5581 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5641 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5701 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5761 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
     5821 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gtcgaccgcc
     5881 aattcaatat ggcgtatatg gactcatgcc aattcaatat ggtggatctg gacctgtgcc
     5941 aattcaatat ggcgtatatg gactcgtgcc aattcaatat ggtggatctg gaccccagcc
     6001 aattcaatat ggcggacttg gcaccatgcc aattcaatat ggcggacctg gcactgtgcc
     6061 aactggggag gggtctactt ggcacggtgc caagtttgag gaggggtctt ggccctgtgc
     6121 caagtccgcc atattgaatt ggcatggtgc caataatggc ggccatattg gctatatgcc
     6181 aggatcaata tataggcaat atccaatatg gccctatgcc aatatggcta ttggccaggt
     6241 tcaatactat gtattggccc tatgccatat agtattccat atatgggttt tcctattgac
     6301 gtagatagcc cctcccaatg ggcggtccca tataccatat atggggcttc ctaataccgc
     6361 ccatagccac tcccccattg acgtcaatgg tctctatata tggtctttcc tattgacgtc
     6421 atatgggcgg tcctattgac gtatatggcg cctcccccat tgacgtcaat tacggtaaat
     6481 ggcccgcctg gctcaatgcc cattgacgtc aataggacca cccaccattg acgtcaatgg
     6541 gatggctcat tgcccattca tatccgttct cacgccccct attgacgtca atgacggtaa
     6601 atggcccact tggcagtaca tcaatatcta ttaatagtaa cttggcaagt acattactat
     6661 tggaagtacg ccagggtaca ttggcagtac tcccattgac gtcaatggcg gtaaatggcc
     6721 cgcgatggct gccaagtaca tccccattga cgtcaatggg gaggggcaat gacgcaaatg
     6781 ggcgttccat tgacgtaaat gggcggtagg cgtgcctaat gggaggtcta tataagcaat
     6841 gctcgtttag ggaac
//
