LOCUS       sequence-180589-        8904 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FLAG-HA-AGO2 (S831E).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8904)
  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 8904)
  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-S831E
FEATURES             Location/Qualifiers
     source          1..8904
                     /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..4577
                     /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     4510..4529
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4598..5197
                     /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(4598..4617)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5094..5114
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5297..5811
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5973..5989
                     /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(5997..6003,6004..6021,6022..6027))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6042..6063
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6180..6197)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6351..6939)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6431..6450)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7110..7901,7902..7970))
                     /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     7733..7752
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7971..8075)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8143..8161
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8199..8221)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8321..8340
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8534..8556
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8684..8703
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(8903..8904,1..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 aacatgacag tgctgaagga agccataccg 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 ggggcggggc gggcgcccga aggtcctccg
     4501 gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct
     4561 catctccggg cctttcgacc tgcagcccaa gcttaccatg accgagtaca agcccacggt
     4621 gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc
     4681 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
     4741 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     4801 cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     4861 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     4921 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     4981 cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     5041 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     5101 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     5161 gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg
     5221 cccgaccgaa aggagcgcac gaccccatgc atcgataaaa taaaagattt tatttagtct
     5281 ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag cttaagtaac
     5341 gccattttgc aaggcatgga aaatacataa ctgagaatag agaagttcag atcaaggtta
     5401 ggaacagaga gacagcagaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
     5461 ccggctcagg gccaagaaca gatggtcccc agatgcggtc ccgccctcag cagtttctag
     5521 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
     5581 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
     5641 aaaagagccc acaacccctc actcggcgcg ccagtcctcc gatagactgc gtcgcccggg
     5701 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
     5761 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tgggtaacag
     5821 tttcttgaag ttggagaaca acattctgag ggtaggagtc gaatattaag taatcctgac
     5881 tcaattagcc actgttttga atccacatac tccaatactc ctgaaatagt tcattatgga
     5941 cagcgcagaa agagctgggg agaattgtga aattgttatc cgctcacaat tccacacaac
     6001 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     6061 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     6121 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     6181 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     6241 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     6301 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6361 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6421 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6481 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6541 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6601 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6661 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6721 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6781 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6841 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6901 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6961 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     7021 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     7081 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     7141 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     7201 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     7261 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     7321 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     7381 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7441 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7501 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7561 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7621 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7681 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7741 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7801 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7861 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7921 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7981 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     8041 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     8101 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     8161 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     8221 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     8281 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
     8341 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     8401 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     8461 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     8521 acgccagggt tttcccagtc acgacgttgt aaaacgacgg cgcaaggaat ggtgcatgca
     8581 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     8641 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     8701 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     8761 ggcgattagt ccaatttgtt aaagacagga tatcagtggt ccaggctcta gttttgactc
     8821 aacaatatca ccagctgaag cctatagagt acgagccata gataaaataa aagattttat
     8881 ttagtctcca gaaaaagggg ggaa
//
