LOCUS       sequence-106494-        8001 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses CMV-FLAG-Ago2 (N-term tag) S387E mutant in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8001)
  AUTHORS   Horman SR, Janas MM, Litterst C, Wang B, MacRae IJ, Sever MJ, 
            Morrissey DV, Graves P, Luo B, Umesalma S, Qi HH, Miraglia LJ, 
            Novina CD, Orth AP
  TITLE     Akt-mediated phosphorylation of argonaute 2 downregulates cleavage 
            and upregulates translational repression of MicroRNA targets.
  JOURNAL   Mol Cell. 2013 May 9;50(3):356-67. doi: 
            10.1016/j.molcel.2013.03.015. Epub 2013 Apr 18.
  PUBMED    23603119
REFERENCE   2  (bases 1 to 8001)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2013.03.015"; journalName: "Mol Cell"; date: 
            "2013-05-9- 9"; volume: "50"; issue: "3"; pages: "356-67"
COMMENT     Sequence Label: pcDNA 3.1 (+) FLAG-Ago2 S387E
FEATURES             Location/Qualifiers
     source          1..8001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             935..958
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2897..2906
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3595..3612)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3601..3825
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3871..4299
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3958..3977)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4168..4189
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4308..4328)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4313..4642
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4493..4628
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4555..4574
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4709..5503
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4763..4782)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5373..5392
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5677..5798
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5714..5733)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5768..5787
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5847..5863)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5847..5863)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5860..5882)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5871..5887
                     /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(5895..5901,5902..5919,5920..5925))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5940..5961
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6078..6095)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6249..6834)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6329..6348)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7005..7796,7797..7865))
                     /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     7628..7647
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7866..7970)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcac catggactac aaagacgatg acgacaaggg
      961 atccgaattc ggtgttctct ctgccattcc cgcacttgca cctcctgcgc cgccgccccc
     1021 catccaagga tatgccttca agcctccacc tagacccgac tttgggacct ccgggagaac
     1081 aatcaaatta caggccaatt tcttcgaaat ggacatcccc aaaattgaca tctatcatta
     1141 tgaattggat atcaagccag agaagtgccc gaggagagtt aacagggaaa tcgtggaaca
     1201 catggtccag cactttaaaa cacagatctt tggggatcgg aagcccgtgt ttgacggcag
     1261 gaagaatcta tacacagcca tgccccttcc gattgggagg gacaaggtgg agctggaggt
     1321 cacgctgcca ggagaaggca aggatcgcat cttcaaggtg tccatcaagt gggtgtcctg
     1381 cgtgagcttg caggcgttac acgatgcact ttcagggcgg ctgcccagcg tcccttttga
     1441 gacgatccag gccctggacg tggtcatgag gcacttgcca tccatgaggt acacccccgt
     1501 gggccgctcc ttcttcaccg cgtccgaagg ctgctctaac cctcttggcg ggggccgaga
     1561 agtgtggttt ggcttccatc agtccgtccg gccttctctc tggaaaatga tgctgaatat
     1621 tgatgtgtca gcaacagcgt tttacaaggc acagccagta atcgagtttg tttgtgaagt
     1681 tttggatttt aaaagtattg aagaacaaca aaaacctctg acagattccc aaagggtaaa
     1741 gtttaccaaa gaaattaaag gtctaaaggt ggagataacg cactgtgggc agatgaagag
     1801 gaagtaccgt gtctgcaatg tgacccggcg gcccgccagt caccaaacat tcccgctgca
     1861 gcaggagagc gggcagacgg tggagtgcac ggtggcccag tatttcaagg acaggcacaa
     1921 gttggttctg cgctaccccc acctcccatg tttacaagtc ggacaggagc agaaacacac
     1981 ctaccttccc ctggaggtct gtaacattgt ggcaggacaa agatgtatta aaaaattaac
     2041 ggacaatcag acctcaacca tgatcagagc gactgctagg tcggcgcccg atcggcaaga
     2101 agagattagc aaattgatgc gaagtgcaga gttcaacaca gatccatacg tccgtgaatt
     2161 tggaatcatg gtcaaagatg agatgacaga cgtgactggg cgggtgctgc agccgccctc
     2221 catcctctac gggggcagga ataaagctat tgcgacccct gtccagggcg tctgggacat
     2281 gcggaacaag cagttccaca cgggcatcga gatcaaggtg tgggccattg cgtgcttcgc
     2341 cccccagcgc cagtgcacgg aagtccatct gaagtccttc acagagcagc tcagaaagat
     2401 ctcgagagac gccggcatgc ccatccaggg ccagccgtgc ttctgcaaat acgcgcaggg
     2461 ggcggacagc gtggagccca tgttccggca cctgaagaac acgtatgcgg gcctgcagct
     2521 ggtggtggtc atcctgcccg gcaagacgcc cgtgtacgcc gaggtcaagc gcgtgggaga
     2581 cacggtgctg gggatggcca cgcagtgcgt gcagatgaag aacgtgcaga ggaccacgcc
     2641 acagaccctg tccaacctct gcctgaagat caacgtcaag ctgggaggcg tgaacaacat
     2701 cctgctgccc cagggcaggc cgccggtgtt ccagcagccc gtcatctttc tgggagcaga
     2761 cgtcactcac ccccccgccg gggatgggaa gaagccctcc attgccgccg tggtgggcag
     2821 catggacgcc caccccaatc gctactgcgc caccgtgcgc gtgcagcagc accggcagga
     2881 gatcatacaa gacctggccg ccatggtccg cgagctcctc atccagttct acaagtccac
     2941 gcgcttcaag cccacccgca tcatcttcta ccgcgacggt gtctctgaag gccagttcca
     3001 gcaggttctc caccacgagt tgctggccat ccgtgaggcc tgtatcaagc tagaaaaaga
     3061 ctaccagccc gggatcacct tcatcgtggt gcagaagagg caccacaccc ggctcttctg
     3121 cactgacaag aacgagcggg ttgggaaaag tggaaacatt ccagcaggca cgactgtgga
     3181 cacgaaaatc acccacccca ccgagttcga cttctacctg tgtagtcacg ctggcatcca
     3241 ggggacaagc aggccttcgc actatcacgt cctctgggac gacaatcgtt tctcctctga
     3301 tgagctgcag atcctaacct accagctgtg tcacacctac gtgcgctgca cacgctccgt
     3361 gtccatccca gcgccagcat actacgctca cctggtggcc ttccgggcca ggtaccacct
     3421 ggtggataag gaacatgaca gtgctgaagg aagccatacc tctgggcaga gtaacgggcg
     3481 agaccaccaa gcactggcca aggcggtcca ggttcaccaa gacactctgc gcaccatgta
     3541 ctttgcttga gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga
     3601 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     3661 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3721 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3781 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     3841 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     3901 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3961 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4021 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4081 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4141 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4201 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     4261 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     4321 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4381 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     4441 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     4501 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     4561 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     4621 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     4681 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     4741 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     4801 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     4861 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     4921 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     4981 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     5041 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     5101 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     5161 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     5221 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     5281 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     5341 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     5401 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     5461 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     5521 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     5581 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     5641 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     5701 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5761 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     5821 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5881 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5941 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     6001 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     6061 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     6121 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     6181 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     6241 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     6301 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     6361 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     6421 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     6481 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6541 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6601 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6661 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     6721 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6781 agcggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6841 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6901 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6961 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     7021 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     7081 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     7141 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     7201 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     7261 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     7321 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     7381 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     7441 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     7501 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7561 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     7621 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     7681 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7741 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7801 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7861 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7921 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7981 cgaaaagtgc cacctgacgt c
//
