LOCUS       sequence-384113-        5410 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 5410)
  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 5410)
  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 EGFP nLAP -1
FEATURES             Location/Qualifiers
     source          1..5410
                     /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             530..1552
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             530..1552
                     /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             1559..1615
                     /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"
     primer_bind     complement(1922..1941)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2360..2383
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2423..2467
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             2480..2500
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2516..2539
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2630..2650)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2630..2648)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2669..2685)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2669..2685)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2682..2704)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2693..2709
                     /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(2717..2723,2724..2741,2742..2747))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2762..2783
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2900..2917)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3071..3659)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3151..3170)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3830..4621,4622..4690))
                     /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     4453..4472
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4691..4795)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4822..5277
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4909..4928)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5119..5140
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5403..5410,1..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 ctaccctgga gaggttcccg acatacctaa agtaccacca tgaaaaagcc
      541 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
      601 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
      661 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
      721 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
      781 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
      841 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
      901 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
      961 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     1021 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     1081 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     1141 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     1201 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     1261 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     1321 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     1381 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     1441 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     1501 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aacgcggagc
     1561 cacgaacttc tctctgttaa agcaagcagg agatgttgaa gaaaaccccg ggcccatggt
     1621 gtccaagggc gaggaactgt tcaccggcgt ggtgcccatc ctggtggagc tggacggcga
     1681 cgtgaacggc cacaagttca gcgtgtccgg cgagggcgaa ggcgacgcca cctacggcaa
     1741 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctggt
     1801 gaccaccctg acctacggcg tgcagtgctt cagcagatac cccgaccaca tgaagcagca
     1861 cgatttcttc aagagcgcca tgcccgaggg ctacgtccag gagcgcacca tctttttcaa
     1921 ggacgacggc aactacaaga ccagggccga ggtgaagttc gagggcgaca cactggtgaa
     1981 caggatcgag ctgaagggca tcgacttcaa agaggacggc aacatcctgg gccacaagct
     2041 ggaatacaac tacaacagcc acaacgtgta cattatggcc gacaagcaga agaacggcat
     2101 caaggtcaac ttcaagatcc ggcacaacat cgaggacggc agcgtgcagc tggccgacca
     2161 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2221 gagcacccag agcgctctca gcaaggaccc caacgagaag cgggaccaca tggtgctgaa
     2281 agaatttgtg acagccgccg gaatcaccct gggcatggac gagctgtaca agagcagcgg
     2341 ccccagcggc agcagcagcc tggaagtgct gttccagggc cccctgagca gctctggccc
     2401 ctctggcagc gccgccaagt tcaaagaaac agctgctgct aaattcgagc ggcagcacat
     2461 ggacagcgga ggaggaggcg agaacctgta ctttcaggga ggcggaggcg gaggggacta
     2521 taaggacgac gacgacaagc tgaccgacga cctgcccagg gcctcgccct ggagaggttc
     2581 ccgacatacc agatgtctcg agggggggcc cggtacccag cttttgttcc ctttagtgag
     2641 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     2701 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     2761 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2821 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2881 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     2941 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3001 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3061 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3121 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3181 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3241 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3301 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3361 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3421 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     3481 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     3541 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     3601 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     3661 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     3721 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     3781 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3841 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3901 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3961 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4021 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4081 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4141 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4201 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4261 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4321 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4381 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     4441 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     4501 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4561 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4621 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     4681 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     4741 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     4801 ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4861 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4921 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4981 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     5041 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5101 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     5161 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     5221 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttcca
     5281 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     5341 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
     5401 ttcccagtca
//
