LOCUS       sequence-180667-        8158 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Lambda-N-HA-AGO2 [S824E, S828E, T830E, S831E, S834E 
            (5xE)].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8158)
  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 8158)
  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: pcDNA3.1+_LN-HA-AGO2-5xE
FEATURES             Location/Qualifiers
     source          1..8158
                     /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             961..1026
                     /codon_start=1
                     /product="N-terminal RNA-binding domain from the lambda 
                     bacteriophage antiterminator protein N (Baron-Benhamou et 
                     al., 2004)"
                     /label=lambda N peptide
                     /translation="MDAQTRRRERRAEKQAQWKAAN"
     CDS             1063..1089
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      3025..3034
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3749..3766)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3755..3979
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4025..4453
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4112..4131)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4322..4343
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4462..4482)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4467..4796
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4647..4782
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4709..4728
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4863..5657
                     /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(4917..4936)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5527..5546
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5831..5952
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5868..5887)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5922..5941
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6001..6017)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6001..6017)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6014..6036)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6025..6041
                     /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(6049..6055,6056..6073,6074..6079))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6094..6115
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6232..6249)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6403..6991)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6483..6502)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7162..7953,7954..8022))
                     /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     7785..7804
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8023..8127)
                     /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 ccgagctcgg atccactagt ccagtgtggt ggaattcacc
      961 atggacgcac aaacacgacg acgtgagcgt cgcgctgaga aacaagctca atggaaagct
     1021 gcaaacccac cgctcgaggc tgcccaagct tacaccatgg cctaccctta tgacgtgccc
     1081 gattacgcta gcggccgcat gtactcggga gccggccccg cacttgcacc tcctgcgccg
     1141 ccgcccccca tccaaggata tgccttcaag cctccaccta gacccgactt tgggacctcc
     1201 gggagaacaa tcaaattaca ggccaatttc ttcgaaatgg acatccccaa aattgacatc
     1261 tatcattatg aattggatat caagccagag aagtgcccga ggagagttaa cagggaaatc
     1321 gtggaacaca tggtccagca ctttaaaaca cagatctttg gggatcggaa gcccgtgttt
     1381 gacggcagga agaatctata cacagccatg ccccttccga ttgggaggga caaggtggag
     1441 ctggaggtca cgctgccagg agaaggcaag gatcgcatct tcaaggtgtc catcaagtgg
     1501 gtgtcctgcg tgagcttgca ggcgttacac gatgcacttt cagggcggct gcccagcgtc
     1561 ccttttgaga cgatccaggc cctggacgtg gtcatgaggc acttgccatc catgaggtac
     1621 acccccgtgg gccgctcctt cttcaccgcg tccgaaggct gctctaaccc tcttggcggg
     1681 ggccgagaag tgtggtttgg cttccatcag tccgtccggc cttctctctg gaaaatgatg
     1741 ctgaatattg atgtgtcagc aacagcgttt tacaaggcac agccagtaat cgagtttgtt
     1801 tgtgaagttt tggattttaa aagtattgaa gaacaacaaa aacctctgac agattcccaa
     1861 agggtaaagt ttaccaaaga aattaaaggt ctaaaggtgg agataacgca ctgtgggcag
     1921 atgaagagga agtaccgtgt ctgcaatgtg acccggcggc ccgccagtca ccaaacattc
     1981 ccgctgcagc aggagagcgg gcagacggtg gagtgcacgg tggcccagta tttcaaggac
     2041 aggcacaagt tggttctgcg ctacccccac ctcccatgtt tacaagtcgg acaggagcag
     2101 aaacacacct accttcccct ggaggtctgt aacattgtgg caggacaaag atgtattaaa
     2161 aaattaacgg acaatcagac ctcaaccatg atcagagcga ctgctaggtc ggcgcccgat
     2221 cggcaagaag agattagcaa attgatgcga agtgcaagtt tcaacacaga tccatacgtc
     2281 cgtgaatttg gaatcatggt caaagatgag atgacagacg tgactgggcg ggtgctgcag
     2341 ccgccctcca tcctctacgg gggcaggaat aaagctattg cgacccctgt ccagggcgtc
     2401 tgggacatgc ggaacaagca gttccacacg ggcatcgaga tcaaggtgtg ggccattgcg
     2461 tgcttcgccc cccagcgcca gtgcacggaa gtccatctga agtccttcac agagcagctc
     2521 agaaagatct cgagagacgc cggcatgccc atccagggcc agccgtgctt ctgcaaatac
     2581 gcgcaggggg cggacagcgt ggagcccatg ttccggcacc tgaagaacac gtatgcgggc
     2641 ctgcagctgg tggtggtcat cctgcccggc aagacgcccg tgtacgccga ggtcaagcgc
     2701 gtgggagaca cggtgctggg gatggccacg cagtgcgtgc agatgaagaa cgtgcagagg
     2761 accacgccac agaccctgtc caacctctgc ctgaagatca acgtcaagct gggaggcgtg
     2821 aacaacatcc tgctgcccca gggcaggccg ccggtgttcc agcagcccgt catctttctg
     2881 ggagcagacg tcactcaccc ccccgccggg gatgggaaga agccctccat tgccgccgtg
     2941 gtgggcagca tggacgccca ccccaatcgc tactgcgcca ccgtgcgcgt gcagcagcac
     3001 cggcaggaga tcatacaaga cctggccgcc atggtccgcg agctcctcat ccagttctac
     3061 aagtccacgc gcttcaagcc cacccgcatc atcttctacc gcgacggtgt ctctgaaggc
     3121 cagttccagc aggttctcca ccacgagttg ctggccatcc gtgaggcctg tatcaagcta
     3181 gaaaaagact accagcccgg gatcaccttc atcgtggtgc agaagaggca ccacacccgg
     3241 ctcttctgca ctgacaagaa cgagcgggtt gggaaaagtg gaaacattcc agcaggcacg
     3301 actgtggaca cgaaaatcac ccaccccacc gagttcgact tctacctgtg tagtcacgct
     3361 ggcatccagg ggacaagcag gccttcgcac tatcacgtcc tctgggacga caatcgtttc
     3421 tcctctgatg agctgcagat cctaacctac cagctgtgtc acacctacgt gcgctgcaca
     3481 cgctccgtgt ccatcccagc gccagcatac tacgctcacc tggtggcctt ccgggccagg
     3541 taccacctgg tggataagga acatgacgag gctgaaggag agcatgagga ggggcaggag
     3601 aacgggcgag accaccaagc actggccaag gcggtccagg ttcaccaaga cactctgcgc
     3661 accatgtact ttgcttgaga attctgcaga tatccagcac agtggcggcc gctcgagtct
     3721 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     3781 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     3841 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     3901 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     3961 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     4021 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4081 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     4141 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4201 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     4261 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     4321 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     4381 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     4441 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     4501 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     4561 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     4621 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     4681 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     4741 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     4801 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     4861 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     4921 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     4981 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     5041 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     5101 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     5161 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     5221 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     5281 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     5341 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     5401 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     5461 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     5521 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     5581 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     5641 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     5701 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     5761 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     5821 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5881 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5941 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     6001 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     6061 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     6121 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     6181 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     6241 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     6301 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     6361 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     6421 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6481 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6541 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6601 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6661 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6721 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6781 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6841 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6901 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6961 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     7021 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     7081 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     7141 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     7201 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     7261 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     7321 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     7381 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     7441 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     7501 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7561 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7621 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7681 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7741 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7801 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7861 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7921 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7981 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     8041 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     8101 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
