LOCUS       sequence-384055-        5391 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 5391)
  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 5391)
  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 +2
FEATURES             Location/Qualifiers
     source          1..5391
                     /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             531..1553
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             531..1553
                     /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             1560..1616
                     /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             1617..2315
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             2343..2366
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2406..2450
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             2463..2483
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2499..2522
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2611..2631)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2611..2629)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2650..2666)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2650..2666)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2663..2685)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2674..2690
                     /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(2698..2704,2705..2722,2723..2728))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2743..2764
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2881..2898)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3052..3640)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3132..3151)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3811..4602,4603..4671))
                     /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     4434..4453
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4672..4776)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4803..5258
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4890..4909)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5100..5121
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5384..5391,1..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 ctacttaggt atgtcgggaa cctctccagg gagtaccacc atgaaaaagc
      541 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
      601 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
      661 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
      721 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
      781 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
      841 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
      901 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
      961 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
     1021 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
     1081 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
     1141 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
     1201 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
     1261 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
     1321 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     1381 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     1441 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     1501 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaacgcggag
     1561 ccacgaactt ctctctgtta aagcaagcag gagatgttga agaaaacccc gggcccatga
     1621 gcgagctgat taaggagaac atgcacatga agctgtacat ggagggcacc gtggacaacc
     1681 atcacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga
     1741 gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctactagct
     1801 tcctctacgg cagcaagacc ttcatcaacc acacccaggg catccccgac ttcttcaagc
     1861 agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc
     1921 tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca
     1981 gaggggtgaa cttcacatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg
     2041 ccttcaccga gacgctgtac cccgctgacg gcggcctgga aggcagaaac gacatggccc
     2101 tgaagctcgt gggcgggagc catctgatcg caaacatcaa gaccacatat agatccaaga
     2161 aacccgctaa gaacctcaag atgcctggcg tctactatgt ggactacaga ctggaaagaa
     2221 tcaaggaggc caacaacgag acctacgtcg agcagcacga ggtggcagtg gccagatact
     2281 gcgacctccc tagcaaactg gggcacaagc ttaatagcag cggccccagc ggcagcagca
     2341 gcctggaagt gctgttccag ggccccctga gcagctctgg cccctctggc agcgccgcca
     2401 agttcaaaga aacagctgct gctaaattcg agcggcagca catggacagc ggaggaggag
     2461 gcgagaacct gtactttcag ggaggcggag gcggagggga ctataaggac gacgacgaca
     2521 agctgaccga cgacctgccc agggcctcgg gtatgtcggg aacctctcca gggatgtctc
     2581 gagggggggc ccggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     2641 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     2701 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     2761 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2821 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     2881 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     2941 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3001 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3061 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3121 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3181 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3241 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3301 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3361 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3421 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3481 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3541 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3601 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3661 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3721 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3781 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3841 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     3901 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     3961 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4021 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4081 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4141 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4201 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4261 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4321 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4381 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4441 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4501 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4561 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4621 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4681 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     4741 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     4801 tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4861 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4921 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4981 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     5041 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     5101 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     5161 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     5221 taacgcgaat tttaacaaaa tattaacgct tacaatttcc attcgccatt caggctgcgc
     5281 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     5341 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc a
//
