LOCUS       sequence-176765-        8947 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FLAG-HA-AGO2 (S824E).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8947)
  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 8947)
  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
FEATURES             Location/Qualifiers
     source          1..8947
                     /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"
     primer_bind     complement(5992..6008)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5992..6008)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6005..6027)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6016..6032
                     /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(6040..6046,6047..6064,6065..6070))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6085..6106
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6223..6240)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6394..6982)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6474..6493)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7153..7944,7945..8013))
                     /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     7776..7795
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8014..8118)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8186..8204
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8242..8264)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8364..8383
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8577..8599
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8727..8746
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(8946..8947,1..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 agccatacct ctgggcagag 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 aagagctggg gagaattaat tcgtaatcat ggtcatagct
     6001 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     6061 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     6121 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6181 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     6241 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     6301 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     6361 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     6421 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     6481 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6541 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     6601 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6661 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6721 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6781 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     6841 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6901 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6961 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     7021 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     7081 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     7141 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     7201 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     7261 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     7321 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     7381 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7441 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7501 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7561 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7621 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7681 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7741 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7801 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7861 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7921 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7981 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     8041 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     8101 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     8161 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     8221 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     8281 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     8341 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     8401 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc
     8461 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     8521 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
     8581 gtcacgacgt tgtaaaacga cggcgcaagg aatggtgcat gcaaggagat ggcgcccaac
     8641 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     8701 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     8761 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggcgatt agtccaattt
     8821 gttaaagaca ggatatcagt ggtccaggct ctagttttga ctcaacaata tcaccagctg
     8881 aagcctatag agtacgagcc atagataaaa taaaagattt tatttagtct ccagaaaaag
     8941 gggggaa
//
