LOCUS       sequence-168653-        6748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Flag tag on the N-terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6748)
  AUTHORS   Khin NC, Lowe JL, Jensen LM, Burgio G
  TITLE     No evidence for genome editing in mouse zygotes and HEK293T human 
            cell line using the DNA-guided Natronobacterium gregoryi Argonaute 
            (NgAgo).
  JOURNAL   PLoS One. 2017 Jun 13;12(6):e0178768. doi: 
            10.1371/journal.pone.0178768. eCollection 2017.
  PUBMED    28609472
REFERENCE   2  (bases 1 to 6748)
  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: "PLoS One.";
            date: "2017-06-13"; volume: "12(6):e0178768. doi"; pages: " 
            10.1371/journal.pone.0178768. eCollection 2017"
COMMENT     Sequence Label: Flag-nls-NgAgo-GK
FEATURES             Location/Qualifiers
     source          1..6748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      628..637
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             637..702
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             709..729
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             733..3390
                     /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    3536..3657
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3573..3592)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3627..3646
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3664..4119)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3801..3822)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4013..4032
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4146..4250
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4252..4609
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4252..4443
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4275..4295)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4460..4595
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4644..5438
                     /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             4644..5438
                     /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(4698..4717)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5308..5327
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5626..5645)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5670..5717
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6046..6634
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6535..6554
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttgcc accatggact ataaggacca cgacggagac
      661 tacaaggatc atgatattga ttacaaagac gatgacgata agatggtgcc aaaaaagaag
      721 agaaaggtag ccacagtgat tgacctcgat tcgaccacca ccgcagacga actgacatcg
      781 ggacacacgt acgacatctc agtcacgctc accggtgtct acgataacac cgacgagcag
      841 catcctcgca tgtctctcgc attcgagcag gacaacggcg agcggcgtta cattaccctg
      901 tggaagaaca cgacacccaa ggatgtcttt acatacgact acgccacggg ctcgacgtac
      961 atcttcacta acatcgacta cgaagtgaag gacggctacg agaatctgac tgcaacatac
     1021 cagacgaccg tcgagaacgc taccgctcag gaagtcggga cgactgacga ggacgaaacg
     1081 ttcgcgggcg gcgagccgct cgaccatcac ttggacgacg cgctcaatga gacgccagac
     1141 gacgcggaga cagagagcga ctcaggccat gtgatgacct cgttcgcctc ccgcgaccaa
     1201 ctccctgagt ggacgctgca tacgtatacg ctaacagcca cagacggcgc aaagacggac
     1261 acggagtacg cgcgacgaac cctcgcatac acggtacggc aggaactcta taccgaccat
     1321 gatgcggctc cggttgcaac tgacgggcta atgcttctca cgccagagcc gctcggcgag
     1381 accccgcttg acctcgattg cggtgtccgg gtcgaggcgg acgagactcg gacactcgat
     1441 tacaccacgg ccaaagaccg gttactcgcc cgcgaactcg tcgaagaggg gctcaaacgc
     1501 tccctctggg atgactacct cgttcgcggc atcgatgaag tcctctcaaa ggagcctgtg
     1561 ctgacttgcg atgagttcga cctacatgag cggtatgacc tctctgtcga agtcggtcac
     1621 agtgggcggg cgtaccttca catcaacttc cgccaccggt tcgtaccgaa gctgacgctc
     1681 gcagacatcg atgatgacaa catctatcct gggctccggg tgaagacgac gtatcgcccc
     1741 cggcgaggac atatcgtctg gggtctgcgg gacgagtgcg ccaccgactc gctcaacacg
     1801 ctgggaaacc agtccgtcgt tgcataccac cgcaacaatc agacacctat taacactgac
     1861 ctcctcgacg ctatcgaggc cgctgaccgg cgagtcgtcg aaacccgacg tcaagggcac
     1921 ggcgatgatg ctgtctcatt cccccaagaa ctgcttgcgg tcgaaccgaa tacgcaccaa
     1981 attaagcagt tcgcctccga cggattccac caacaggccc gctcaaagac gcgtctctcg
     2041 gcctcccgct gcagcgagaa agcgcaagcg ttcgccgagc ggcttgaccc ggtgcgtctc
     2101 aatgggtcca cggtagagtt ctcctcggag tttttcaccg ggaacaacga gcagcaactg
     2161 cgcctcctct acgagaacgg tgagtcggtt ctgacgttcc gcgacggggc gcgtggtgcg
     2221 caccccgacg agacattctc gaaaggtatc gtcaatccac cagagtcgtt cgaggtggcc
     2281 gtagtactgc ccgagcagca ggcagatacc tgcaaagcgc agtgggacac gatggctgac
     2341 ctcctcaacc aagctggcgc gccaccgaca cggagcgaga ccgtccaata tgatgcgttc
     2401 tcctcgccag agagcatcag cctcaatgtg gctggagcca tcgaccctag cgaggtagac
     2461 gcggcattcg tcgtactgcc gccggaccaa gaaggattcg cagacctcgc cagtccgaca
     2521 gagacgtacg acgagctgaa gaaggcgctt gccaacatgg gcatttacag ccagatggcg
     2581 tacttcgacc ggttccgcga cgcgaaaata ttctatactc gtaacgtggc actcgggctg
     2641 ctggcagccg ctggcggcgt cgcattcaca accgaacatg cgatgcctgg ggacgcagat
     2701 atgttcattg ggattgatgt ctctcggagc taccccgagg acggtgccag cggccagata
     2761 aacattgccg cgacggcgac cgccgtctac aaggatggaa ctatcctcgg ccactcgtcc
     2821 acccgaccgc agctcgggga gaaactacag tcgacggatg ttcgtgacat tatgaagaat
     2881 gccatcctcg gctaccagca ggtgaccggt gagtcgccga cccatatcgt catccaccgt
     2941 gatggcttca tgaacgaaga cctcgacccc gccacggaat tcctcaacga acaaggcgtc
     3001 gagtacgaca tcgtcgaaat ccgcaagcag ccccagacac gcctgctggc agtctccgat
     3061 gtgcagtacg atacgcctgt gaagagcatc gccgctatca accagaacga gccacgggca
     3121 acggtcgcca ccttcggcgc acccgaatac ttagcgacac gcgatggagg cggccttccc
     3181 cgcccaatcc aaattgaacg agtcgccggc gaaaccgaca tcgagacgct cactcgccaa
     3241 gtctatctgc tctcccagtc gcatatccag gtccataact cgactgcgcg cctacccatc
     3301 accaccgcat acgccgacca ggcaagtact cacgcgacca agggttacct cgtccagacc
     3361 ggagcgttcg agtctaatgt cggattcctc cgggatccat atgtaagtaa gtaagcggcc
     3421 gcgactctag atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     3481 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     3541 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3601 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg
     3661 cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     3721 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     3781 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     3841 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     3901 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     3961 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     4021 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     4081 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa
     4141 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4201 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg
     4261 aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     4321 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     4381 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     4441 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     4501 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     4561 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag
     4621 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     4681 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     4741 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     4801 tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga
     4861 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     4921 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     4981 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     5041 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     5101 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     5161 ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     5221 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     5281 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     5341 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     5401 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     5461 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     5521 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     5581 ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga
     5641 gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg
     5701 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
     5761 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
     5821 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
     5881 agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5941 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6001 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6061 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6121 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6181 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6241 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6301 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6361 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6421 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6481 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6541 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6601 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6661 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     6721 tgtggataac cgtattaccg ccatgcat
//
