LOCUS       sequence-273732-        8071 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses human AGO2 with an N-terminal 3XFLAG tag in mammalian 
            cells (using a CMV promoter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8071)
  AUTHORS   McGeary SE, Lin KS, Shi CY, Pham TM, Bisaria N, Kelley GM, Bartel DP
  TITLE     The biochemical basis of microRNA targeting efficacy.
  JOURNAL   Science. 2019 Dec 20;366(6472). pii: science.aav1741. doi: 
            10.1126/science.aav1741. Epub 2019 Dec 5.
  PUBMED    31806698
REFERENCE   2  (bases 1 to 8071)
  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"; journalName: "Science."; 
            date: "2019-12-20"; volume: "366(6472). pii: science.aav1741. doi"; 
            pages: " 10.1126/science.aav1741. Epub 2019 Dec 5"
COMMENT     Sequence Label: pcDNA3.3-3XFLAG-AGO2
FEATURES             Location/Qualifiers
     source          1..8071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        128..507
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        508..711
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     662..682
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     708..732
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      820..829
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             829..894
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      2830..2839
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3535..3554)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3579..3627
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3829..4257
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3916..3935)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4126..4147
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4266..4286)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4271..4600
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4451..4586
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4513..4532
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4667..5461
                     /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(4721..4740)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5331..5350
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5637..5758
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5674..5693)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5728..5747
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5807..5823)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5807..5823)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5820..5842)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5831..5847
                     /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(5855..5861,5862..5879,5880..5885))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5900..5921
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6038..6055)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6209..6797)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6289..6308)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6968..7759,7760..7828))
                     /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     7591..7610
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7829..7933)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8008..8027)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
       61 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
      121 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
      181 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      241 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      301 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      361 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      421 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      481 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
      541 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
      601 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
      661 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg
      721 aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg
      781 gaccgatcca gcctccggac tctagaggat cgaacccttg ccaccatgga ctacaaagac
      841 catgacggtg attataaaga tcatgatatc gattacaagg atgacgatga caagcttggt
      901 ggttcttact cgggagccgg ccccgcactt gcacctcctg cgccgccgcc ccccatccaa
      961 ggatatgcct tcaagcctcc acctagaccc gactttggga cctccgggag aacaatcaaa
     1021 ttacaggcca atttcttcga aatggacatc cccaaaattg acatctatca ttatgaattg
     1081 gatatcaagc cagagaagtg cccgaggaga gttaacaggg aaatcgtgga acacatggtc
     1141 cagcacttta aaacacagat ctttggggat cggaagcccg tgtttgacgg caggaagaat
     1201 ctatacacag ccatgcccct tccgattggg agggacaagg tggagctgga ggtcacgctg
     1261 ccaggagaag gcaaggatcg catcttcaag gtgtccatca agtgggtgtc ctgcgtgagc
     1321 ttgcaggcgt tacacgatgc actttcaggg cggctgccta gcgtcccttt tgagacgatc
     1381 caggccctgg acgtggtcat gaggcacttg ccatccatga ggtacacccc cgtgggccgc
     1441 tccttcttca ccgcgtccga aggctgctct aaccctcttg gcgggggccg agaagtgtgg
     1501 tttggcttcc atcagtccgt ccggccttct ctctggaaaa tgatgctgaa tattgatgtg
     1561 tcagcaacag cgttttacaa ggcacagcca gtaatcgagt ttgtttgtga agttttggat
     1621 tttaaaagta ttgaagaaca acaaaaacct ctgacagatt cccaaagggt aaagtttacc
     1681 aaagaaatta aaggtctaaa ggtggagata acgcactgtg ggcagatgaa gaggaagtac
     1741 cgcgtctgca atgtgacccg gcggcccgcc agtcaccaaa cattcccgct gcagcaggag
     1801 agcgggcaga cggtggagtg cacggtggcc cagtatttca aggacaggca caagcttgtt
     1861 ctgcgctacc cccacctccc atgtttacaa gtcggacagg agcagaaaca cacctacctt
     1921 cccctggagg tctgtaacat tgtggcagga caaagatgta ttaaaaaatt aacggacaat
     1981 cagacctcaa ccatgatcag agcaactgct aggtcggcgc ccgatcggca agaagagatt
     2041 agcaaattga tgcgaagtgc aagtttcaac acagatccat acgtccgtga atttggaatc
     2101 atggtcaaag atgagatgac agacgtgact gggcgggtgc tgcagccgcc ctccatcctc
     2161 tacgggggca ggaataaagc tattgcgacc cctgtccagg gcgtctggga catgcggaac
     2221 aagcagttcc acacgggcat cgagatcaag gtgtgggcca ttgcgtgctt cgccccccag
     2281 cgccagtgca cggaagtcca tctgaagtcc ttcacagagc agctcagaaa gatctcgaga
     2341 gacgctggca tgcccatcca gggccagccg tgcttctgca aatacgcgca gggggcggac
     2401 agcgtggagc ccatgttccg gcacctgaag aacacgtatg cgggcctgca gctggtggtg
     2461 gtcatcctgc ccggcaagac gcccgtgtac gccgaggtca agcgcgtggg agacacggtg
     2521 ctggggatgg ccacgcagtg cgtgcagatg aagaacgtgc agaggaccac gccacagacc
     2581 ctgtccaacc tctgcctgaa gatcaacgtc aagctgggag gcgtgaacaa catcctgctg
     2641 ccccagggca ggccgccggt gttccagcag cccgtcatct ttctgggagc agacgtcact
     2701 cacccccccg ccggggatgg gaagaagccc tccattgccg ccgtggtggg cagcatggac
     2761 gcccacccca atcgctactg cgccaccgtg cgcgtgcagc agcaccggca ggagatcata
     2821 caagacctgg ccgccatggt ccgcgagctc ctcatccagt tctacaagtc cacgcgcttc
     2881 aagcccaccc gcatcatctt ctaccgcgac ggtgtctctg aaggccagtt ccagcaggtt
     2941 ctccaccacg agttgctggc catccgtgag gcctgtatca agctagaaaa agactaccag
     3001 cccgggatca ccttcatcgt ggtgcagaag aggcaccaca cccggctctt ctgcactgac
     3061 aagaacgagc gggttgggaa aagtggaaac attccagcag gcacgactgt ggacacgaaa
     3121 atcacccacc ccaccgagtt cgacttctac ctgtgtagtc acgctggcat ccaggggaca
     3181 agcaggcctt cgcactatca cgtcctctgg gacgacaatc gtttctcctc tgatgagctg
     3241 cagatcctaa cctaccagct gtgtcacacc tacgtgcgct gcacacgctc cgtgtccatc
     3301 ccagcgccag catactacgc tcacctggtg gccttccggg ccaggtacca cctggtggat
     3361 aaggaacatg acagtgctga aggaagccat acctctgggc agagtaacgg gcgagaccac
     3421 caagcactgg ccaaggcggt ccaggttcac caagacactc tgcgcaccat gtactttgct
     3481 tgaaagggtt cgatccctac cggttagtaa tgagtttaaa cgggggaggc taactgaaac
     3541 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     3601 aacgcacggg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     3661 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     3721 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     3781 gccctgccat agcagatctg cgcagctggg gctctagggg gtatccccac gcgccctgta
     3841 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3901 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3961 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4021 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     4081 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4141 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     4201 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     4261 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     4321 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     4381 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     4441 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4501 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     4561 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     4621 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     4681 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     4741 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     4801 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     4861 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4921 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4981 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     5041 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     5101 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     5161 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     5221 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     5281 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     5341 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     5401 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     5461 agcgggactc tggggttcgc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     5521 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     5581 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     5641 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5701 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5761 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5821 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5881 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5941 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6001 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     6061 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     6121 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6181 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6241 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6301 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6361 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6421 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6481 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6541 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6601 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6661 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6721 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6781 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6841 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6901 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6961 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     7021 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     7081 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     7141 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     7201 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7261 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7321 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     7381 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7441 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7501 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7561 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7621 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7681 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7741 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7801 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7861 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7921 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     7981 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     8041 gtatctgctc cctgcttgtg tgttggaggt c
//
