LOCUS       sequence-158751-        6679 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of NgAgo (Argonaute from N. gregoryi)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6679)
  AUTHORS   Gao F, Shen XZ, Jiang F, Wu Y, Han C
  TITLE     DNA-guided genome editing using the Natronobacterium gregoryi 
            Argonaute.
  JOURNAL   Nat Biotechnol. 2016 May 2. doi: 10.1038/nbt.3547.
  PUBMED    27136078
REFERENCE   2  (bases 1 to 6679)
  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"; journalName: "Nat 
            Biotechnol. 2016 May 2. doi: 10.1038/nbt.3547."
COMMENT     Sequence Label: nls-NgAgo-GK
FEATURES             Location/Qualifiers
     source          1..6679
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        16..319
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        320..523
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     474..494
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      583..592
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             595..615
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             619..3276
                     /codon_start=1
                     /product="Argonaute protein from Natronobacterium gregoryi 
                     (Gao et al., 2016)"
                     /label=NgAgo
                     /note="reportedly a DNA-guided endonuclease that does not 
                     require a PAM site, although this interpretation has been 
                     challenged (Cyranoski, 2016)"
                     /translation="TVIDLDSTTTADELTSGHTYDISVTLTGVYDNTDEQHPRMSLAFE
                     QDNGERRYITLWKNTTPKDVFTYDYATGSTYIFTNIDYEVKDGYENLTATYQTTVENAT
                     AQEVGTTDEDETFAGGEPLDHHLDDALNETPDDAETESDSGHVMTSFASRDQLPEWTLH
                     TYTLTATDGAKTDTEYARRTLAYTVRQELYTDHDAAPVATDGLMLLTPEPLGETPLDLD
                     CGVRVEADETRTLDYTTAKDRLLARELVEEGLKRSLWDDYLVRGIDEVLSKEPVLTCDE
                     FDLHERYDLSVEVGHSGRAYLHINFRHRFVPKLTLADIDDDNIYPGLRVKTTYRPRRGH
                     IVWGLRDECATDSLNTLGNQSVVAYHRNNQTPINTDLLDAIEAADRRVVETRRQGHGDD
                     AVSFPQELLAVEPNTHQIKQFASDGFHQQARSKTRLSASRCSEKAQAFAERLDPVRLNG
                     STVEFSSEFFTGNNEQQLRLLYENGESVLTFRDGARGAHPDETFSKGIVNPPESFEVAV
                     VLPEQQADTCKAQWDTMADLLNQAGAPPTRSETVQYDAFSSPESISLNVAGAIDPSEVD
                     AAFVVLPPDQEGFADLASPTETYDELKKALANMGIYSQMAYFDRFRDAKIFYTRNVALG
                     LLAAAGGVAFTTEHAMPGDADMFIGIDVSRSYPEDGASGQINIAATATAVYKDGTILGH
                     SSTRPQLGEKLQSTDVRDIMKNAILGYQQVTGESPTHIVIHRDGFMNEDLDPATEFLNE
                     QGVEYDIVEIRKQPQTRLLAVSDVQYDTPVKSIAAINQNEPRATVATFGAPEYLATRDG
                     GGLPRPIQIERVAGETDIETLTRQVYLLSQSHIQVHNSTARLPITTAYADQASTHATKG
                     YLVQTGAFESNVGFL"
     polyA_signal    3422..3543
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3459..3478)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3513..3532
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3550..4005)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3687..3708)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3899..3918
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4032..4136
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4138..4495
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4138..4329
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4161..4181)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4346..4481
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4530..5324
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4530..5324
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4584..4603)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5194..5213
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5512..5531)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5556..5603
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5932..6520
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6421..6440
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
       61 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      121 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      181 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      241 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      301 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      361 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      421 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      481 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
      541 gatccgctag cgctaccgga ctcagatctc gagctcaagc ttgccaccat ggtgccaaaa
      601 aagaagagaa aggtagccac agtgattgac ctcgattcga ccaccaccgc agacgaactg
      661 acatcgggac acacgtacga catctcagtc acgctcaccg gtgtctacga taacaccgac
      721 gagcagcatc ctcgcatgtc tctcgcattc gagcaggaca acggcgagcg gcgttacatt
      781 accctgtgga agaacacgac acccaaggat gtctttacat acgactacgc cacgggctcg
      841 acgtacatct tcactaacat cgactacgaa gtgaaggacg gctacgagaa tctgactgca
      901 acataccaga cgaccgtcga gaacgctacc gctcaggaag tcgggacgac tgacgaggac
      961 gaaacgttcg cgggcggcga gccgctcgac catcacttgg acgacgcgct caatgagacg
     1021 ccagacgacg cggagacaga gagcgactca ggccatgtga tgacctcgtt cgcctcccgc
     1081 gaccaactcc ctgagtggac gctgcatacg tatacgctaa cagccacaga cggcgcaaag
     1141 acggacacgg agtacgcgcg acgaaccctc gcatacacgg tacggcagga actctatacc
     1201 gaccatgatg cggctccggt tgcaactgac gggctaatgc ttctcacgcc agagccgctc
     1261 ggcgagaccc cgcttgacct cgattgcggt gtccgggtcg aggcggacga gactcggaca
     1321 ctcgattaca ccacggccaa agaccggtta ctcgcccgcg aactcgtcga agaggggctc
     1381 aaacgctccc tctgggatga ctacctcgtt cgcggcatcg atgaagtcct ctcaaaggag
     1441 cctgtgctga cttgcgatga gttcgaccta catgagcggt atgacctctc tgtcgaagtc
     1501 ggtcacagtg ggcgggcgta ccttcacatc aacttccgcc accggttcgt accgaagctg
     1561 acgctcgcag acatcgatga tgacaacatc tatcctgggc tccgggtgaa gacgacgtat
     1621 cgcccccggc gaggacatat cgtctggggt ctgcgggacg agtgcgccac cgactcgctc
     1681 aacacgctgg gaaaccagtc cgtcgttgca taccaccgca acaatcagac acctattaac
     1741 actgacctcc tcgacgctat cgaggccgct gaccggcgag tcgtcgaaac ccgacgtcaa
     1801 gggcacggcg atgatgctgt ctcattcccc caagaactgc ttgcggtcga accgaatacg
     1861 caccaaatta agcagttcgc ctccgacgga ttccaccaac aggcccgctc aaagacgcgt
     1921 ctctcggcct cccgctgcag cgagaaagcg caagcgttcg ccgagcggct tgacccggtg
     1981 cgtctcaatg ggtccacggt agagttctcc tcggagtttt tcaccgggaa caacgagcag
     2041 caactgcgcc tcctctacga gaacggtgag tcggttctga cgttccgcga cggggcgcgt
     2101 ggtgcgcacc ccgacgagac attctcgaaa ggtatcgtca atccaccaga gtcgttcgag
     2161 gtggccgtag tactgcccga gcagcaggca gatacctgca aagcgcagtg ggacacgatg
     2221 gctgacctcc tcaaccaagc tggcgcgcca ccgacacgga gcgagaccgt ccaatatgat
     2281 gcgttctcct cgccagagag catcagcctc aatgtggctg gagccatcga ccctagcgag
     2341 gtagacgcgg cattcgtcgt actgccgccg gaccaagaag gattcgcaga cctcgccagt
     2401 ccgacagaga cgtacgacga gctgaagaag gcgcttgcca acatgggcat ttacagccag
     2461 atggcgtact tcgaccggtt ccgcgacgcg aaaatattct atactcgtaa cgtggcactc
     2521 gggctgctgg cagccgctgg cggcgtcgca ttcacaaccg aacatgcgat gcctggggac
     2581 gcagatatgt tcattgggat tgatgtctct cggagctacc ccgaggacgg tgccagcggc
     2641 cagataaaca ttgccgcgac ggcgaccgcc gtctacaagg atggaactat cctcggccac
     2701 tcgtccaccc gaccgcagct cggggagaaa ctacagtcga cggatgttcg tgacattatg
     2761 aagaatgcca tcctcggcta ccagcaggtg accggtgagt cgccgaccca tatcgtcatc
     2821 caccgtgatg gcttcatgaa cgaagacctc gaccccgcca cggaattcct caacgaacaa
     2881 ggcgtcgagt acgacatcgt cgaaatccgc aagcagcccc agacacgcct gctggcagtc
     2941 tccgatgtgc agtacgatac gcctgtgaag agcatcgccg ctatcaacca gaacgagcca
     3001 cgggcaacgg tcgccacctt cggcgcaccc gaatacttag cgacacgcga tggaggcggc
     3061 cttccccgcc caatccaaat tgaacgagtc gccggcgaaa ccgacatcga gacgctcact
     3121 cgccaagtct atctgctctc ccagtcgcat atccaggtcc ataactcgac tgcgcgccta
     3181 cccatcacca ccgcatacgc cgaccaggca agtactcacg cgaccaaggg ttacctcgtc
     3241 cagaccggag cgttcgagtc taatgtcgga ttcctccggg atccatatgt aagtaagtaa
     3301 gcggccgcga ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt
     3361 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     3421 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     3481 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     3541 ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     3601 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     3661 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     3721 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     3781 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     3841 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     3901 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     3961 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     4021 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4081 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     4141 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     4201 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     4261 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4321 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     4381 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     4441 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     4501 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     4561 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     4621 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     4681 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     4741 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     4801 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     4861 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     4921 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     4981 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     5041 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     5101 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     5161 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     5221 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     5281 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     5341 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     5401 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     5461 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     5521 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     5581 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     5641 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     5701 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     5761 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
     5821 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5881 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5941 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6001 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6061 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6121 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6181 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6241 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6301 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6361 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6421 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6481 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6541 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     6601 tgattctgtg gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg
     6661 ggtcattagt tcatagccc
//
