LOCUS       sequence-407047-        5220 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 5220)
  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 5220)
  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 +2
FEATURES             Location/Qualifiers
     source          1..5220
                     /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             547..570
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             586..606
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             622..666
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             703..726
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1060..1079)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1477..1533
                     /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(2440..2460)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2440..2458)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2479..2495)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2479..2495)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2492..2514)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2503..2519
                     /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(2527..2533,2534..2551,2552..2557))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2572..2593
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2710..2727)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2881..3469)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2961..2980)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3640..4431,4432..4500))
                     /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     4263..4282
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4501..4605)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4632..5087
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4719..4738)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4929..4950
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5213..5220,1..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 ctacaggtat gtcgggaacc tctccaggac tgaccgacga cctgcccagg
      541 gcctcggact ataaggacga cgacgacaag ggcggaggcg gaggggagaa cctgtacttt
      601 cagggaagcg gaggaggagg caaagaaaca gctgctgcta aattcgagcg gcagcacatg
      661 gacagctctg gcccctctgg cagcgccgcc aagttcagca gcctggaagt gctgttccag
      721 ggccccctga gcagcagcgg ccccagcggc agcatggtgt ccaagggcga ggaactgttc
      781 accggcgtgg tgcccatcct ggtggagctg gacggcgacg tgaacggcca caagttcagc
      841 gtgtccggcg agggcgaagg cgacgccacc tacggcaagc tgaccctgaa gttcatctgc
      901 accaccggca agctgcccgt gccctggccc accctggtga ccaccctgac ctacggcgtg
      961 cagtgcttca gcagataccc cgaccacatg aagcagcacg atttcttcaa gagcgccatg
     1021 cccgagggct acgtccagga gcgcaccatc tttttcaagg acgacggcaa ctacaagacc
     1081 agggccgagg tgaagttcga gggcgacaca ctggtgaaca ggatcgagct gaagggcatc
     1141 gacttcaaag aggacggcaa catcctgggc cacaagctgg aatacaacta caacagccac
     1201 aacgtgtaca ttatggccga caagcagaag aacggcatca aggtcaactt caagatccgg
     1261 cacaacatcg aggacggcag cgtgcagctg gccgaccact accagcagaa cacccccatc
     1321 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagag cgctctcagc
     1381 aaggacccca acgagaagcg ggaccacatg gtgctgaaag aatttgtgac agccgccgga
     1441 atcaccctgg gcatggacga gctgtacaag cgcggagcca cgaacttctc tctgttaaag
     1501 caagcaggag atgttgaaga aaaccccggg cccatggggg caggtgccac cggccgcgcc
     1561 atggacgggc cgcgcctgct gctgttgctg cttctggggg tgtcccttgg aggtgccaag
     1621 gaggcatgcc ccacaggcct gtacacacac agcggtgagt gctgcaaagc ctgcaacctg
     1681 ggcgagggtg tggcccagcc ttgtggagcc aaccagaccg tgtgtgagcc ctgcctggac
     1741 agcgtgacgt tctccgacgt ggtgagcgcg accgagccgt gcaagccgtg caccgagtgc
     1801 gtggggctcc agagcatgtc ggcgccgtgc gtggaggccg acgacgccgt gtgccgctgc
     1861 gcctacggct actaccagga tgagacgact gggcgctgcg aggcgtgccg cgtgtgcgag
     1921 gcgggctcgg gcctcgtgtt ctcctgccag gacaagcaga acaccgtgtg cgaggagtgc
     1981 cccgacggca cgtattccga cgaggccaac cacgtggacc cgtgcctgcc ctgcaccgtg
     2041 tgcgaggaca ccgagcgcca gctccgcgag tgcacacgct gggccgacgc cgagtgcgag
     2101 gagatccctg gccgttggat tacacggtcc acacccccag agggctcgga cagcacagcc
     2161 cccagcaccc aggagcctga ggcacctcca gaacaagacc tcatagccag cacggtggca
     2221 ggtgtggtga ccacagtgat gggcagctcc cagcccgtgg tgacccgagg caccaccgac
     2281 aacctcatcc ctgtctattg ctccatcctg gctgctgtgg ttgtgggcct tgtggcctac
     2341 atagccttca agaggtggaa cagggggatc ctctgacctg gtatgtcggg aacctctcca
     2401 gggattctcg agggggggcc cggtacccag cttttgttcc ctttagtgag ggttaattgc
     2461 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     2521 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     2581 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2641 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     2701 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     2761 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     2821 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2881 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     2941 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3001 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3061 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3121 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3181 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3241 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3301 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     3361 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3421 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3481 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     3541 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     3601 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     3661 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     3721 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     3781 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     3841 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     3901 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     3961 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     4021 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     4081 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     4141 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     4201 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     4261 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     4321 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     4381 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     4441 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     4501 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     4561 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     4621 agtgccacct gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4681 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4741 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4801 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4861 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4921 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4981 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     5041 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc
     5101 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     5161 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
//
