LOCUS       sequence-180644-        8916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FLAG-HA-AGO2 (S824E-S828E-S831E).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8916)
  AUTHORS   Golden RJ, Chen B, Li T, Braun J, Manjunath H, Chen X, Wu J, Schmid 
            V, Chang TC, Kopp F, Ramirez-Martinez A, Tagliabracci VS, Chen ZJ, 
            Xie Y, Mendell JT
  TITLE     An Argonaute phosphorylation cycle promotes microRNA-mediated 
            silencing.
  JOURNAL   Nature. 2017 Feb 9;542(7640):197-202. doi: 10.1038/nature21025. Epub
            2017 Jan 23.
  PUBMED    28114302
REFERENCE   2  (bases 1 to 8916)
  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: 
            "10.1038/nature21025"; journalName: "Nature"; date: "2017-02-9- 9"; 
            volume: "542"; issue: "7640"; pages: "197-202"
COMMENT     Sequence Label: MSCV-Puro-FH-AGO2-S824E-S828E-S831E
FEATURES             Location/Qualifiers
     source          1..8916
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1440..1463
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1464..1490
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      3426..3435
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        4090..4589
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4125..4148)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4522..4541
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4610..5209
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4610..4629)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5106..5126
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5309..5823
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5985..6001
                     /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(6009..6015,6016..6033,6034..6039))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6054..6075
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6192..6209)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6363..6951)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6443..6462)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7122..7913,7914..7982))
                     /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     7745..7764
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7983..8087)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8155..8173
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8211..8233)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8333..8352
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8546..8568
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8696..8715
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(8915..8916,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatctctcga ggttaacgaa ttcgccatgg
     1441 actacaagga cgacgatgac aagtaccctt atgacgtgcc cgattacgct agcggccgca
     1501 tgtactcggg agccggcccc gcacttgcac ctcctgcgcc gccgcccccc atccaaggat
     1561 atgccttcaa gcctccacct agacccgact ttgggacctc cgggagaaca atcaaattac
     1621 aggccaattt cttcgaaatg gacatcccca aaattgacat ctatcattat gaattggata
     1681 tcaagccaga gaagtgcccg aggagagtta acagggaaat cgtggaacac atggtccagc
     1741 actttaaaac acagatcttt ggggatcgga agcccgtgtt tgacggcagg aagaatctat
     1801 acacagccat gccccttccg attgggaggg acaaggtgga gctggaggtc acgctgccag
     1861 gagaaggcaa ggatcgcatc ttcaaggtgt ccatcaagtg ggtgtcctgc gtgagcttgc
     1921 aggcgttaca cgatgcactt tcagggcggc tgcccagcgt cccttttgag acgatccagg
     1981 ccctggacgt ggtcatgagg cacttgccat ccatgaggta cacccccgtg ggccgctcct
     2041 tcttcaccgc gtccgaaggc tgctctaacc ctcttggcgg gggccgagaa gtgtggtttg
     2101 gcttccatca gtccgtccgg ccttctctct ggaaaatgat gctgaatatt gatgtgtcag
     2161 caacagcgtt ttacaaggca cagccagtaa tcgagtttgt ttgtgaagtt ttggatttta
     2221 aaagtattga agaacaacaa aaacctctga cagattccca aagggtaaag tttaccaaag
     2281 aaattaaagg tctaaaggtg gagataacgc actgtgggca gatgaagagg aagtaccgtg
     2341 tctgcaatgt gacccggcgg cccgccagtc accaaacatt cccgctgcag caggagagcg
     2401 ggcagacggt ggagtgcacg gtggcccagt atttcaagga caggcacaag ttggttctgc
     2461 gctaccccca cctcccatgt ttacaagtcg gacaggagca gaaacacacc taccttcccc
     2521 tggaggtctg taacattgtg gcaggacaaa gatgtattaa aaaattaacg gacaatcaga
     2581 cctcaaccat gatcagagcg actgctaggt cggcgcccga tcggcaagaa gagattagca
     2641 aattgatgcg aagtgcaagt ttcaacacag atccatacgt ccgtgaattt ggaatcatgg
     2701 tcaaagatga gatgacagac gtgactgggc gggtgctgca gccgccctcc atcctctacg
     2761 ggggcaggaa taaagctatt gcgacccctg tccagggcgt ctgggacatg cggaacaagc
     2821 agttccacac gggcatcgag atcaaggtgt gggccattgc gtgcttcgcc ccccagcgcc
     2881 agtgcacgga agtccatctg aagtccttca cagagcagct cagaaagatc tcgagagacg
     2941 ccggcatgcc catccagggc cagccgtgct tctgcaaata cgcgcagggg gcggacagcg
     3001 tggagcccat gttccggcac ctgaagaaca cgtatgcggg cctgcagctg gtggtggtca
     3061 tcctgcccgg caagacgccc gtgtacgccg aggtcaagcg cgtgggagac acggtgctgg
     3121 ggatggccac gcagtgcgtg cagatgaaga acgtgcagag gaccacgcca cagaccctgt
     3181 ccaacctctg cctgaagatc aacgtcaagc tgggaggcgt gaacaacatc ctgctgcccc
     3241 agggcaggcc gccggtgttc cagcagcccg tcatctttct gggagcagac gtcactcacc
     3301 cccccgccgg ggatgggaag aagccctcca ttgccgccgt ggtgggcagc atggacgccc
     3361 accccaatcg ctactgcgcc accgtgcgcg tgcagcagca ccggcaggag atcatacaag
     3421 acctggccgc catggtccgc gagctcctca tccagttcta caagtccacg cgcttcaagc
     3481 ccacccgcat catcttctac cgcgacggtg tctctgaagg ccagttccag caggttctcc
     3541 accacgagtt gctggccatc cgtgaggcct gtatcaagct agaaaaagac taccagcccg
     3601 ggatcacctt catcgtggtg cagaagaggc accacacccg gctcttctgc actgacaaga
     3661 acgagcgggt tgggaaaagt ggaaacattc cagcaggcac gactgtggac acgaaaatca
     3721 cccaccccac cgagttcgac ttctacctgt gtagtcacgc tggcatccag gggacaagca
     3781 ggccttcgca ctatcacgtc ctctgggacg acaatcgttt ctcctctgat gagctgcaga
     3841 tcctaaccta ccagctgtgt cacacctacg tgcgctgcac acgctccgtg tccatcccag
     3901 cgccagcata ctacgctcac ctggtggcct tccgggccag gtaccacctg gtggataagg
     3961 aacatgacga ggctgaagga gagcataccg aggggcagag taacgggcga gaccaccaag
     4021 cactggccaa ggcggtccag gttcaccaag acactctgcg caccatgtac tttgcttgag
     4081 aattctaccg ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag
     4141 ccccgctggg cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca
     4201 ccggtaggcg ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc
     4261 ccctagtcag gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg
     4321 aagtagcacg tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg
     4381 taggcctttg gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc
     4441 tgggaagggg tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc
     4501 gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt
     4561 tctcctcttc ctcatctccg ggcctttcga cctgcagccc aagcttacca tgaccgagta
     4621 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
     4681 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
     4741 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     4801 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     4861 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     4921 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     4981 cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     5041 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     5101 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     5161 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca
     5221 cgacccgcag cgcccgaccg aaaggagcgc acgaccccat gcatcgataa aataaaagat
     5281 tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt ttggcaagct
     5341 agcttaagta acgccatttt gcaaggcatg gaaaatacat aactgagaat agagaagttc
     5401 agatcaaggt taggaacaga gagacagcag aatatgggcc aaacaggata tctgtggtaa
     5461 gcagttcctg ccccggctca gggccaagaa cagatggtcc ccagatgcgg tcccgccctc
     5521 agcagtttct agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct
     5581 gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc
     5641 cccgagctca ataaaagagc ccacaacccc tcactcggcg cgccagtcct ccgatagact
     5701 gcgtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt
     5761 ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt
     5821 catgggtaac agtttcttga agttggagaa caacattctg agggtaggag tcgaatatta
     5881 agtaatcctg actcaattag ccactgtttt gaatccacat actccaatac tcctgaaata
     5941 gttcattatg gacagcgcag aaagagctgg ggagaattgt gaaattgtta tccgctcaca
     6001 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     6061 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     6121 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     6181 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     6241 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     6301 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6361 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6421 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6481 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6541 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6601 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6661 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6721 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6781 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     6841 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6901 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6961 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     7021 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     7081 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     7141 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     7201 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     7261 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     7321 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7381 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     7441 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7501 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     7561 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     7621 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     7681 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     7741 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     7801 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     7861 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7921 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7981 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     8041 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     8101 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     8161 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     8221 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     8281 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     8341 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg
     8401 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     8461 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     8521 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggcgcaagga
     8581 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     8641 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     8701 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     8761 gtccggcgta gaggcgatta gtccaatttg ttaaagacag gatatcagtg gtccaggctc
     8821 tagttttgac tcaacaatat caccagctga agcctataga gtacgagcca tagataaaat
     8881 aaaagatttt atttagtctc cagaaaaagg ggggaa
//
