LOCUS       sequence-180111-        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        115..418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        419..622
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     573..593
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      682..691
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             691..756
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             763..783
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             787..3444
                     /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    3590..3711
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3627..3646)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3681..3700
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3718..4173)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3855..3876)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4067..4086
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4200..4304
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4306..4663
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4306..4497
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4329..4349)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4514..4649
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4698..5492
                     /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             4698..5492
                     /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(4752..4771)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5362..5381
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5680..5699)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5724..5771
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6100..6688
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6589..6608
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta
       61 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      121 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      181 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      241 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      301 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      361 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      421 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      481 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      541 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      601 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc gctaccggac
      661 tcagatctcg agctcaagct tgccaccatg gactataagg accacgacgg agactacaag
      721 gatcatgata ttgattacaa agacgatgac gataagatgg tgccaaaaaa gaagagaaag
      781 gtagccacag tgattgacct cgattcgacc accaccgcag acgaactgac atcgggacac
      841 acgtacgaca tctcagtcac gctcaccggt gtctacgata acaccgacga gcagcatcct
      901 cgcatgtctc tcgcattcga gcaggacaac ggcgagcggc gttacattac cctgtggaag
      961 aacacgacac ccaaggatgt ctttacatac gactacgcca cgggctcgac gtacatcttc
     1021 actaacatcg actacgaagt gaaggacggc tacgagaatc tgactgcaac ataccagacg
     1081 accgtcgaga acgctaccgc tcaggaagtc gggacgactg acgaggacga aacgttcgcg
     1141 ggcggcgagc cgctcgacca tcacttggac gacgcgctca atgagacgcc agacgacgcg
     1201 gagacagaga gcgactcagg ccatgtgatg acctcgttcg cctcccgcga ccaactccct
     1261 gagtggacgc tgcatacgta tacgctaaca gccacagacg gcgcaaagac ggacacggag
     1321 tacgcgcgac gaaccctcgc atacacggta cggcaggaac tctataccga ccatgatgcg
     1381 gctccggttg caactgacgg gctaatgctt ctcacgccag agccgctcgg cgagaccccg
     1441 cttgacctcg attgcggtgt ccgggtcgag gcggacgaga ctcggacact cgattacacc
     1501 acggccaaag accggttact cgcccgcgaa ctcgtcgaag aggggctcaa acgctccctc
     1561 tgggatgact acctcgttcg cggcatcgat gaagtcctct caaaggagcc tgtgctgact
     1621 tgcgatgagt tcgacctaca tgagcggtat gacctctctg tcgaagtcgg tcacagtggg
     1681 cgggcgtacc ttcacatcaa cttccgccac cggttcgtac cgaagctgac gctcgcagac
     1741 atcgatgatg acaacatcta tcctgggctc cgggtgaaga cgacgtatcg cccccggcga
     1801 ggacatatcg tctggggtct gcgggacgag tgcgccaccg actcgctcaa cacgctggga
     1861 aaccagtccg tcgttgcata ccaccgcaac aatcagacac ctattaacac tgacctcctc
     1921 gacgctatcg aggccgctga ccggcgagtc gtcgaaaccc gacgtcaagg gcacggcgat
     1981 gatgctgtct cattccccca agaactgctt gcggtcgaac cgaatacgca ccaaattaag
     2041 cagttcgcct ccgacggatt ccaccaacag gcccgctcaa agacgcgtct ctcggcctcc
     2101 cgctgcagcg agaaagcgca agcgttcgcc gagcggcttg acccggtgcg tctcaatggg
     2161 tccacggtag agttctcctc ggagtttttc accgggaaca acgagcagca actgcgcctc
     2221 ctctacgaga acggtgagtc ggttctgacg ttccgcgacg gggcgcgtgg tgcgcacccc
     2281 gacgagacat tctcgaaagg tatcgtcaat ccaccagagt cgttcgaggt ggccgtagta
     2341 ctgcccgagc agcaggcaga tacctgcaaa gcgcagtggg acacgatggc tgacctcctc
     2401 aaccaagctg gcgcgccacc gacacggagc gagaccgtcc aatatgatgc gttctcctcg
     2461 ccagagagca tcagcctcaa tgtggctgga gccatcgacc ctagcgaggt agacgcggca
     2521 ttcgtcgtac tgccgccgga ccaagaagga ttcgcagacc tcgccagtcc gacagagacg
     2581 tacgacgagc tgaagaaggc gcttgccaac atgggcattt acagccagat ggcgtacttc
     2641 gaccggttcc gcgacgcgaa aatattctat actcgtaacg tggcactcgg gctgctggca
     2701 gccgctggcg gcgtcgcatt cacaaccgaa catgcgatgc ctggggacgc agatatgttc
     2761 attgggattg atgtctctcg gagctacccc gaggacggtg ccagcggcca gataaacatt
     2821 gccgcgacgg cgaccgccgt ctacaaggat ggaactatcc tcggccactc gtccacccga
     2881 ccgcagctcg gggagaaact acagtcgacg gatgttcgtg acattatgaa gaatgccatc
     2941 ctcggctacc agcaggtgac cggtgagtcg ccgacccata tcgtcatcca ccgtgatggc
     3001 ttcatgaacg aagacctcga ccccgccacg gaattcctca acgaacaagg cgtcgagtac
     3061 gacatcgtcg aaatccgcaa gcagccccag acacgcctgc tggcagtctc cgatgtgcag
     3121 tacgatacgc ctgtgaagag catcgccgct atcaaccaga acgagccacg ggcaacggtc
     3181 gccaccttcg gcgcacccga atacttagcg acacgcgatg gaggcggcct tccccgccca
     3241 atccaaattg aacgagtcgc cggcgaaacc gacatcgaga cgctcactcg ccaagtctat
     3301 ctgctctccc agtcgcatat ccaggtccat aactcgactg cgcgcctacc catcaccacc
     3361 gcatacgccg accaggcaag tactcacgcg accaagggtt acctcgtcca gaccggagcg
     3421 ttcgagtcta atgtcggatt cctccgggat ccatatgtaa gtaagtaagc ggccgcgact
     3481 ctagatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     3541 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     3601 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     3661 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa
     3721 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     3781 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     3841 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     3901 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     3961 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     4021 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     4081 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     4141 ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc
     4201 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4261 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa
     4321 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     4381 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     4441 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     4501 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4561 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     4621 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag
     4681 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     4741 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     4801 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     4861 gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     4921 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     4981 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     5041 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     5101 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     5161 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     5221 aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     5281 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     5341 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     5401 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     5461 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     5521 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     5581 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     5641 catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat
     5701 accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg
     5761 gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc
     5821 gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag
     5881 ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc
     5941 aggttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6001 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6061 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6121 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6181 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6241 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6301 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6361 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6421 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6481 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6541 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6601 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6661 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6721 ggccttttgc tggccttttg ctcacatg
//
