LOCUS       sequence-384066-        5227 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 5227)
  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 5227)
  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 -1
FEATURES             Location/Qualifiers
     source          1..5227
                     /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             1376..1432
                     /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(1739..1758)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2177..2200
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2240..2284
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             2297..2317
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2333..2356
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2447..2467)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2447..2465)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2486..2502)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2486..2502)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2499..2521)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2510..2526
                     /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(2534..2540,2541..2558,2559..2564))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2579..2600
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2717..2734)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2888..3476)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2968..2987)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3647..4438,4439..4507))
                     /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     4270..4289
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4508..4612)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4639..5094
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4726..4745)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4936..4957
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5220..5227,1..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 ctaccctgga gaggttcccg acatacctaa agtaccacca tgggggcagg
      541 tgccaccggc cgcgccatgg acgggccgcg cctgctgctg ttgctgcttc tgggggtgtc
      601 ccttggaggt gccaaggagg catgccccac aggcctgtac acacacagcg gtgagtgctg
      661 caaagcctgc aacctgggcg agggtgtggc ccagccttgt ggagccaacc agaccgtgtg
      721 tgagccctgc ctggacagcg tgacgttctc cgacgtggtg agcgcgaccg agccgtgcaa
      781 gccgtgcacc gagtgcgtgg ggctccagag catgtcggcg ccgtgcgtgg aggccgacga
      841 cgccgtgtgc cgctgcgcct acggctacta ccaggatgag acgactgggc gctgcgaggc
      901 gtgccgcgtg tgcgaggcgg gctcgggcct cgtgttctcc tgccaggaca agcagaacac
      961 cgtgtgcgag gagtgccccg acggcacgta ttccgacgag gccaaccacg tggacccgtg
     1021 cctgccctgc accgtgtgcg aggacaccga gcgccagctc cgcgagtgca cacgctgggc
     1081 cgacgccgag tgcgaggaga tccctggccg ttggattaca cggtccacac ccccagaggg
     1141 ctcggacagc acagccccca gcacccagga gcctgaggca cctccagaac aagacctcat
     1201 agccagcacg gtggcaggtg tggtgaccac agtgatgggc agctcccagc ccgtggtgac
     1261 ccgaggcacc accgacaacc tcatccctgt ctattgctcc atcctggctg ctgtggttgt
     1321 gggccttgtg gcctacatag ccttcaagag gtggaacagg gggatcctcc gcggagccac
     1381 gaacttctct ctgttaaagc aagcaggaga tgttgaagaa aaccccgggc ccatggtgtc
     1441 caagggcgag gaactgttca ccggcgtggt gcccatcctg gtggagctgg acggcgacgt
     1501 gaacggccac aagttcagcg tgtccggcga gggcgaaggc gacgccacct acggcaagct
     1561 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctggtgac
     1621 caccctgacc tacggcgtgc agtgcttcag cagatacccc gaccacatga agcagcacga
     1681 tttcttcaag agcgccatgc ccgagggcta cgtccaggag cgcaccatct ttttcaagga
     1741 cgacggcaac tacaagacca gggccgaggt gaagttcgag ggcgacacac tggtgaacag
     1801 gatcgagctg aagggcatcg acttcaaaga ggacggcaac atcctgggcc acaagctgga
     1861 atacaactac aacagccaca acgtgtacat tatggccgac aagcagaaga acggcatcaa
     1921 ggtcaacttc aagatccggc acaacatcga ggacggcagc gtgcagctgg ccgaccacta
     1981 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     2041 cacccagagc gctctcagca aggaccccaa cgagaagcgg gaccacatgg tgctgaaaga
     2101 atttgtgaca gccgccggaa tcaccctggg catggacgag ctgtacaaga gcagcggccc
     2161 cagcggcagc agcagcctgg aagtgctgtt ccagggcccc ctgagcagct ctggcccctc
     2221 tggcagcgcc gccaagttca aagaaacagc tgctgctaaa ttcgagcggc agcacatgga
     2281 cagcggagga ggaggcgaga acctgtactt tcagggaggc ggaggcggag gggactataa
     2341 ggacgacgac gacaagctga ccgacgacct gcccagggcc tcgccctgga gaggttcccg
     2401 acataccaga tgtctcgagg gggggcccgg tacccagctt ttgttccctt tagtgagggt
     2461 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     2521 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     2581 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     2641 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     2701 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     2761 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2821 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2881 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2941 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3001 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3061 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3121 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3181 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3241 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3301 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     3361 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3421 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3481 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     3541 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     3601 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     3661 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     3721 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     3781 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3841 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3901 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3961 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4021 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4081 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4141 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4201 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4261 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4321 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4381 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4441 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4501 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     4561 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     4621 cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4681 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     4741 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4801 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     4861 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     4921 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     4981 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5041 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc
     5101 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     5161 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     5221 ccagtca
//
