LOCUS       sequence-384029-        5401 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 5401)
  AUTHORS   Seibert M, Kurrle N, Stolp V, Nurnberger H, Tzschentke S, Borner L, 
            Wempe F, Serve H, Schnutgen F
  TITLE     Selection-free endogenous tagging of cell lines by bicistronic 
            co-expression of the surface antigen NGFR.
  JOURNAL   MethodsX. 2022 Nov 18;9:101929. doi: 10.1016/j.mex.2022.101929. 
            eCollection 2022.
  PUBMED    36444289
REFERENCE   2  (bases 1 to 5401)
  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: "MethodsX.";
            date: "2022-11-18"; volume: "9:101929. doi"; pages: " 
            10.1016/j.mex.2022.101929. eCollection 2022"
COMMENT     Sequence Label: pGTag Hygro EGFP cLAP +1
FEATURES             Location/Qualifiers
     source          1..5401
                     /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             548..571
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             584..604
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             620..664
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             701..724
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1058..1077)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1475..1531
                     /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             1532..2557
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1532..2557
                     /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"
     primer_bind     complement(2621..2641)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2621..2639)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2660..2676)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2660..2676)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2673..2695)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2684..2700
                     /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(2708..2714,2715..2732,2733..2738))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2753..2774
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2891..2908)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3062..3650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3142..3161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3821..4612,4613..4681))
                     /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     4444..4463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4682..4786)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4813..5268
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4900..4919)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5110..5131
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5394..5401,1..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 ataccgtcga ctacgggtat gtcgggaacc tctccaggta ctgaccgacg acctgcccag
      541 ggcctcggac tataaggacg acgacgacaa gggaggcgga ggggagaacc tgtactttca
      601 gggaagcgga ggaggaggca aagaaacagc tgctgctaaa ttcgagcggc agcacatgga
      661 cagctctggc ccctctggca gcgccgccaa gttcagcagc ctggaagtgc tgttccaggg
      721 ccccctgagc agcagcggcc ccagcggcag catggtgtcc aagggcgagg aactgttcac
      781 cggcgtggtg cccatcctgg tggagctgga cggcgacgtg aacggccaca agttcagcgt
      841 gtccggcgag ggcgaaggcg acgccaccta cggcaagctg accctgaagt tcatctgcac
      901 caccggcaag ctgcccgtgc cctggcccac cctggtgacc accctgacct acggcgtgca
      961 gtgcttcagc agataccccg accacatgaa gcagcacgat ttcttcaaga gcgccatgcc
     1021 cgagggctac gtccaggagc gcaccatctt tttcaaggac gacggcaact acaagaccag
     1081 ggccgaggtg aagttcgagg gcgacacact ggtgaacagg atcgagctga agggcatcga
     1141 cttcaaagag gacggcaaca tcctgggcca caagctggaa tacaactaca acagccacaa
     1201 cgtgtacatt atggccgaca agcagaagaa cggcatcaag gtcaacttca agatccggca
     1261 caacatcgag gacggcagcg tgcagctggc cgaccactac cagcagaaca cccccatcgg
     1321 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagagcg ctctcagcaa
     1381 ggaccccaac gagaagcggg accacatggt gctgaaagaa tttgtgacag ccgccggaat
     1441 caccctgggc atggacgagc tgtacaagcg cggagccacg aacttctctc tgttaaagca
     1501 agcaggagat gttgaagaaa accccgggcc catgaaaaag cctgaactca ccgcgacgtc
     1561 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
     1621 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
     1681 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
     1741 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
     1801 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
     1861 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     1921 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     1981 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
     2041 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     2101 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     2161 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     2221 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     2281 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     2341 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     2401 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     2461 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     2521 ccgacgcccc agcactcgtc cgagggcaaa ggaatgactg gtatgtcggg aacctctcca
     2581 gggatgtctc gagggggggc ccggtaccca gcttttgttc cctttagtga gggttaattg
     2641 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     2701 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     2761 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2821 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2881 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2941 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3001 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3061 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3121 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3181 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3241 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3301 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3361 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3421 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3481 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3541 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3601 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3661 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3721 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3781 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3841 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3901 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3961 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4021 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4081 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4141 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4201 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4261 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4321 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4381 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4441 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4501 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4561 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4621 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4681 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4741 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4801 aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4861 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4921 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4981 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     5041 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5101 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     5161 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     5221 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc attcgccatt
     5281 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     5341 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     5401 a
//
