LOCUS       sequence-384068-        5218 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 5218)
  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 5218)
  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 NGFR EGFP cLAP +1
FEATURES             Location/Qualifiers
     source          1..5218
                     /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"
     primer_bind     complement(2438..2458)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2438..2456)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2477..2493)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2477..2493)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2490..2512)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2501..2517
                     /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(2525..2531,2532..2549,2550..2555))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2570..2591
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2708..2725)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2879..3467)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2959..2978)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3638..4429,4430..4498))
                     /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     4261..4280
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4499..4603)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4630..5085
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4717..4736)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4927..4948
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5211..5218,1..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 catgggggca ggtgccaccg gccgcgccat
     1561 ggacgggccg cgcctgctgc tgttgctgct tctgggggtg tcccttggag gtgccaagga
     1621 ggcatgcccc acaggcctgt acacacacag cggtgagtgc tgcaaagcct gcaacctggg
     1681 cgagggtgtg gcccagcctt gtggagccaa ccagaccgtg tgtgagccct gcctggacag
     1741 cgtgacgttc tccgacgtgg tgagcgcgac cgagccgtgc aagccgtgca ccgagtgcgt
     1801 ggggctccag agcatgtcgg cgccgtgcgt ggaggccgac gacgccgtgt gccgctgcgc
     1861 ctacggctac taccaggatg agacgactgg gcgctgcgag gcgtgccgcg tgtgcgaggc
     1921 gggctcgggc ctcgtgttct cctgccagga caagcagaac accgtgtgcg aggagtgccc
     1981 cgacggcacg tattccgacg aggccaacca cgtggacccg tgcctgccct gcaccgtgtg
     2041 cgaggacacc gagcgccagc tccgcgagtg cacacgctgg gccgacgccg agtgcgagga
     2101 gatccctggc cgttggatta cacggtccac acccccagag ggctcggaca gcacagcccc
     2161 cagcacccag gagcctgagg cacctccaga acaagacctc atagccagca cggtggcagg
     2221 tgtggtgacc acagtgatgg gcagctccca gcccgtggtg acccgaggca ccaccgacaa
     2281 cctcatccct gtctattgct ccatcctggc tgctgtggtt gtgggccttg tggcctacat
     2341 agccttcaag aggtggaaca gggggatcct ctgactggta tgtcgggaac ctctccaggg
     2401 atgtctcgag ggggggcccg gtacccagct tttgttccct ttagtgaggg ttaattgcgc
     2461 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2521 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2581 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2641 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     2701 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2761 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2821 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2881 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2941 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3001 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3061 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3121 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3181 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3241 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3301 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3361 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3421 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3481 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3541 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3601 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3661 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3721 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3781 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3841 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3901 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3961 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4021 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4081 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4141 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4201 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4261 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4321 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4381 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4441 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4501 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4561 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4621 tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4681 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     4741 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4801 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     4861 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4921 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     4981 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     5041 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttccatt cgccattcag
     5101 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     5161 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtca
//
