LOCUS       sequence-384051-        5404 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 5404)
  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 5404)
  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 +0
FEATURES             Location/Qualifiers
     source          1..5404
                     /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             549..572
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             588..608
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             624..668
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             705..728
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1062..1081)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1479..1535
                     /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             1536..2561
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1536..2561
                     /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(2624..2644)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2624..2642)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2663..2679)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2663..2679)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2676..2698)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2687..2703
                     /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(2711..2717,2718..2735,2736..2741))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2756..2777
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2894..2911)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3065..3653)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3145..3164)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3824..4615,4616..4684))
                     /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     4447..4466
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4685..4789)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4816..5271
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4903..4922)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5113..5134
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5397..5404,1..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 tctccaggct actgaccgac gacctgccca
      541 gggcctcgga ctataaggac gacgacgaca agggcggagg cggaggggag aacctgtact
      601 ttcagggaag cggaggagga ggcaaagaaa cagctgctgc taaattcgag cggcagcaca
      661 tggacagctc tggcccctct ggcagcgccg ccaagttcag cagcctggaa gtgctgttcc
      721 agggccccct gagcagcagc ggccccagcg gcagcatggt gtccaagggc gaggaactgt
      781 tcaccggcgt ggtgcccatc ctggtggagc tggacggcga cgtgaacggc cacaagttca
      841 gcgtgtccgg cgagggcgaa ggcgacgcca cctacggcaa gctgaccctg aagttcatct
      901 gcaccaccgg caagctgccc gtgccctggc ccaccctggt gaccaccctg acctacggcg
      961 tgcagtgctt cagcagatac cccgaccaca tgaagcagca cgatttcttc aagagcgcca
     1021 tgcccgaggg ctacgtccag gagcgcacca tctttttcaa ggacgacggc aactacaaga
     1081 ccagggccga ggtgaagttc gagggcgaca cactggtgaa caggatcgag ctgaagggca
     1141 tcgacttcaa agaggacggc aacatcctgg gccacaagct ggaatacaac tacaacagcc
     1201 acaacgtgta cattatggcc gacaagcaga agaacggcat caaggtcaac ttcaagatcc
     1261 ggcacaacat cgaggacggc agcgtgcagc tggccgacca ctaccagcag aacaccccca
     1321 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag agcgctctca
     1381 gcaaggaccc caacgagaag cgggaccaca tggtgctgaa agaatttgtg acagccgccg
     1441 gaatcaccct gggcatggac gagctgtaca agcgcggagc cacgaacttc tctctgttaa
     1501 agcaagcagg agatgttgaa gaaaaccccg ggcccatgaa aaagcctgaa ctcaccgcga
     1561 cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct
     1621 cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc
     1681 gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat
     1741 cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct
     1801 attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc
     1861 ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc
     1921 agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg
     1981 atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca
     2041 ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc
     2101 ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg
     2161 gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg
     2221 tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact
     2281 tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca
     2341 ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg
     2401 cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa
     2461 tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg
     2521 gaaaccgacg ccccagcact cgtccgaggg caaaggaatg acggtatgtc gggaacctct
     2581 ccagggatgt ctcgaggggg ggcccggtac ccagcttttg ttccctttag tgagggttaa
     2641 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     2701 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     2761 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     2821 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     2881 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     2941 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     3001 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3061 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3121 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3181 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3241 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3301 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3361 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3421 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3481 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     3541 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3601 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3661 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3721 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3781 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3841 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3901 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3961 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     4021 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4081 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     4141 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4201 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4261 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4321 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4381 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     4441 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4501 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4561 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4621 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4681 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4741 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4801 gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4861 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4921 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4981 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     5041 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5101 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5161 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5221 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt tccattcgcc
     5281 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     5341 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
     5401 gtca
//
