LOCUS       sequence-384112-        5393 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9-mediated generic protein tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5393)
  AUTHORS   Thone FMB, Kurrle NS, von Melchner H, Schnutgen F
  TITLE     CRISPR/Cas9-mediated generic protein tagging in mammalian cells.
  JOURNAL   Methods. 2019 Jul 15;164-165:59-66. doi: 
            10.1016/j.ymeth.2019.02.018. Epub 2019 Feb 22.
  PUBMED    30797903
REFERENCE   2  (bases 1 to 5393)
  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: "Methods."; 
            date: "2019-07-15"; volume: "164-165:59-66. doi"; pages: " 
            10.1016/j.ymeth.2019.02.018. Epub 2019 Feb 22"
COMMENT     Sequence Label: pGTag Hygro BFP nLAP -0
FEATURES             Location/Qualifiers
     source          1..5393
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     7..24
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8..24
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     34..53
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        34..52
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     263..282
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        361..436
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             532..1554
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             532..1554
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1561..1617
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1618..2316
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             2344..2367
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2407..2451
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             2464..2484
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2500..2523
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2613..2633)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2613..2631)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2652..2668)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2652..2668)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2665..2687)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2676..2692
                     /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(2700..2706,2707..2724,2725..2730))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2745..2766
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2883..2900)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3054..3642)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3134..3153)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3813..4604,4605..4673))
                     /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     4436..4455
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4674..4778)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4805..5260
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4892..4911)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5102..5123
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5386..5393,1..15)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg
       61 gagctacccg cattaccctg ttatccctaa gatccgacta tttcccatga ttccttcata
      121 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
      181 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      241 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
      301 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggtatgtcgg gaacctctcc
      361 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
      421 ggcaccgagt cggtgctttt ttgaattcag atcttacatt tcaggacatc tagcttatcg
      481 ataccgtcat ctacccctgg agaggttccc gacataccta aaagtaccac catgaaaaag
      541 cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc
      601 gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg
      661 cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt
      721 tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc
      781 agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg
      841 cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct
      901 gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa
      961 tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa
     1021 actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt
     1081 tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat
     1141 gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg
     1201 gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag
     1261 cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg
     1321 gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc
     1381 gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact
     1441 gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa
     1501 gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaacgcgga
     1561 gccacgaact tctctctgtt aaagcaagca ggagatgttg aagaaaaccc cgggcccatg
     1621 agcgagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac cgtggacaac
     1681 catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac ccagaccatg
     1741 agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct ggctactagc
     1801 ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga cttcttcaag
     1861 cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga cgggggcgtg
     1921 ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa cgtcaagatc
     1981 agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact cggctgggag
     2041 gccttcaccg agacgctgta ccccgctgac ggcggcctgg aaggcagaaa cgacatggcc
     2101 ctgaagctcg tgggcgggag ccatctgatc gcaaacatca agaccacata tagatccaag
     2161 aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag actggaaaga
     2221 atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt ggccagatac
     2281 tgcgacctcc ctagcaaact ggggcacaag cttaatagca gcggccccag cggcagcagc
     2341 agcctggaag tgctgttcca gggccccctg agcagctctg gcccctctgg cagcgccgcc
     2401 aagttcaaag aaacagctgc tgctaaattc gagcggcagc acatggacag cggaggagga
     2461 ggcgagaacc tgtactttca gggaggcgga ggcggagggg actataagga cgacgacgac
     2521 aagctgaccg acgacctgcc cagggcctcg cctggagagg ttcccgacat accagatgtc
     2581 tcgagggggg gcccggtacc cagcttttgt tccctttagt gagggttaat tgcgcgcttg
     2641 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     2701 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     2761 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2821 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2881 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2941 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3001 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3061 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3121 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3181 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3241 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     3301 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3361 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3421 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3481 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3541 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3601 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3661 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3721 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3781 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3841 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3901 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     3961 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4021 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4081 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     4141 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     4201 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     4261 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     4321 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     4381 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     4441 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     4501 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4561 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4621 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4681 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4741 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4801 cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4861 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     4921 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4981 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     5041 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5101 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     5161 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     5221 tttaacgcga attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc
     5281 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     5341 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tca
//
