LOCUS       sequence-384109-        5411 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 5411)
  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 5411)
  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 -0
FEATURES             Location/Qualifiers
     source          1..5411
                     /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"
     primer_bind     complement(1924..1943)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2362..2385
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2425..2469
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             2482..2502
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2518..2541
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2631..2651)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2631..2649)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2670..2686)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2670..2686)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2683..2705)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2694..2710
                     /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(2718..2724,2725..2742,2743..2748))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2763..2784
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2901..2918)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3072..3660)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3152..3171)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3831..4622,4623..4691))
                     /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     4454..4473
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4692..4796)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4823..5278
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4910..4929)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5120..5141
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5404..5411,1..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 gtgtccaagg gcgaggaact gttcaccggc gtggtgccca tcctggtgga gctggacggc
     1681 gacgtgaacg gccacaagtt cagcgtgtcc ggcgagggcg aaggcgacgc cacctacggc
     1741 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctg
     1801 gtgaccaccc tgacctacgg cgtgcagtgc ttcagcagat accccgacca catgaagcag
     1861 cacgatttct tcaagagcgc catgcccgag ggctacgtcc aggagcgcac catctttttc
     1921 aaggacgacg gcaactacaa gaccagggcc gaggtgaagt tcgagggcga cacactggtg
     1981 aacaggatcg agctgaaggg catcgacttc aaagaggacg gcaacatcct gggccacaag
     2041 ctggaataca actacaacag ccacaacgtg tacattatgg ccgacaagca gaagaacggc
     2101 atcaaggtca acttcaagat ccggcacaac atcgaggacg gcagcgtgca gctggccgac
     2161 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2221 ctgagcaccc agagcgctct cagcaaggac cccaacgaga agcgggacca catggtgctg
     2281 aaagaatttg tgacagccgc cggaatcacc ctgggcatgg acgagctgta caagagcagc
     2341 ggccccagcg gcagcagcag cctggaagtg ctgttccagg gccccctgag cagctctggc
     2401 ccctctggca gcgccgccaa gttcaaagaa acagctgctg ctaaattcga gcggcagcac
     2461 atggacagcg gaggaggagg cgagaacctg tactttcagg gaggcggagg cggaggggac
     2521 tataaggacg acgacgacaa gctgaccgac gacctgccca gggcctcgcc tggagaggtt
     2581 cccgacatac cagatgtctc gagggggggc ccggtaccca gcttttgttc cctttagtga
     2641 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     2701 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     2761 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     2821 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2881 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     2941 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3001 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3061 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3121 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3181 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3241 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3301 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3361 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3421 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3481 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3541 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3601 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     3661 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     3721 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     3781 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     3841 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     3901 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     3961 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4021 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4081 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4141 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4201 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4261 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4321 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4381 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4441 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4501 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4561 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4621 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     4681 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     4741 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     4801 tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4861 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     4921 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     4981 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     5041 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5101 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     5161 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     5221 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc
     5281 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     5341 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     5401 tttcccagtc a
//
