LOCUS       sequence-384126-        5228 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 5228)
  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 5228)
  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 nLAP -0
FEATURES             Location/Qualifiers
     source          1..5228
                     /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             1378..1434
                     /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(1741..1760)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2179..2202
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2242..2286
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             2299..2319
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2335..2358
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2448..2468)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2448..2466)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2487..2503)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2487..2503)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2500..2522)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2511..2527
                     /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(2535..2541,2542..2559,2560..2565))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2580..2601
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2718..2735)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2889..3477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2969..2988)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3648..4439,4440..4508))
                     /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     4271..4290
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4509..4613)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4640..5095
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4727..4746)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4937..4958
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5221..5228,1..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 ctacccctgg agaggttccc gacataccta aaagtaccac catgggggca
      541 ggtgccaccg gccgcgccat ggacgggccg cgcctgctgc tgttgctgct tctgggggtg
      601 tcccttggag gtgccaagga ggcatgcccc acaggcctgt acacacacag cggtgagtgc
      661 tgcaaagcct gcaacctggg cgagggtgtg gcccagcctt gtggagccaa ccagaccgtg
      721 tgtgagccct gcctggacag cgtgacgttc tccgacgtgg tgagcgcgac cgagccgtgc
      781 aagccgtgca ccgagtgcgt ggggctccag agcatgtcgg cgccgtgcgt ggaggccgac
      841 gacgccgtgt gccgctgcgc ctacggctac taccaggatg agacgactgg gcgctgcgag
      901 gcgtgccgcg tgtgcgaggc gggctcgggc ctcgtgttct cctgccagga caagcagaac
      961 accgtgtgcg aggagtgccc cgacggcacg tattccgacg aggccaacca cgtggacccg
     1021 tgcctgccct gcaccgtgtg cgaggacacc gagcgccagc tccgcgagtg cacacgctgg
     1081 gccgacgccg agtgcgagga gatccctggc cgttggatta cacggtccac acccccagag
     1141 ggctcggaca gcacagcccc cagcacccag gagcctgagg cacctccaga acaagacctc
     1201 atagccagca cggtggcagg tgtggtgacc acagtgatgg gcagctccca gcccgtggtg
     1261 acccgaggca ccaccgacaa cctcatccct gtctattgct ccatcctggc tgctgtggtt
     1321 gtgggccttg tggcctacat agccttcaag aggtggaaca gggggatcct ccgcggagcc
     1381 acgaacttct ctctgttaaa gcaagcagga gatgttgaag aaaaccccgg gcccatggtg
     1441 tccaagggcg aggaactgtt caccggcgtg gtgcccatcc tggtggagct ggacggcgac
     1501 gtgaacggcc acaagttcag cgtgtccggc gagggcgaag gcgacgccac ctacggcaag
     1561 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctggtg
     1621 accaccctga cctacggcgt gcagtgcttc agcagatacc ccgaccacat gaagcagcac
     1681 gatttcttca agagcgccat gcccgagggc tacgtccagg agcgcaccat ctttttcaag
     1741 gacgacggca actacaagac cagggccgag gtgaagttcg agggcgacac actggtgaac
     1801 aggatcgagc tgaagggcat cgacttcaaa gaggacggca acatcctggg ccacaagctg
     1861 gaatacaact acaacagcca caacgtgtac attatggccg acaagcagaa gaacggcatc
     1921 aaggtcaact tcaagatccg gcacaacatc gaggacggca gcgtgcagct ggccgaccac
     1981 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     2041 agcacccaga gcgctctcag caaggacccc aacgagaagc gggaccacat ggtgctgaaa
     2101 gaatttgtga cagccgccgg aatcaccctg ggcatggacg agctgtacaa gagcagcggc
     2161 cccagcggca gcagcagcct ggaagtgctg ttccagggcc ccctgagcag ctctggcccc
     2221 tctggcagcg ccgccaagtt caaagaaaca gctgctgcta aattcgagcg gcagcacatg
     2281 gacagcggag gaggaggcga gaacctgtac tttcagggag gcggaggcgg aggggactat
     2341 aaggacgacg acgacaagct gaccgacgac ctgcccaggg cctcgcctgg agaggttccc
     2401 gacataccag atgtctcgag ggggggcccg gtacccagct tttgttccct ttagtgaggg
     2461 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     2521 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     2581 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     2641 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2701 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2761 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2821 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2881 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2941 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3001 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3061 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3121 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3181 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3241 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3301 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3361 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3421 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3481 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3541 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3601 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3661 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3721 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3781 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3841 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3901 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3961 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4021 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4081 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4141 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4201 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4261 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4321 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4381 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4441 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4501 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4561 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     4621 ccccgaaaag tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4681 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     4741 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4801 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     4861 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4921 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     4981 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     5041 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttccatt
     5101 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     5161 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     5221 cccagtca
//
