LOCUS       sequence-384132-        5226 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 5226)
  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 5226)
  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 +2
FEATURES             Location/Qualifiers
     source          1..5226
                     /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             1377..1433
                     /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(1740..1759)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2178..2201
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2241..2285
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             2298..2318
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2334..2357
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2446..2466)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2446..2464)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2485..2501)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2485..2501)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2498..2520)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2509..2525
                     /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(2533..2539,2540..2557,2558..2563))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2578..2599
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2716..2733)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2887..3475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2967..2986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3646..4437,4438..4506))
                     /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     4269..4288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4507..4611)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4638..5093
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4725..4744)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4935..4956
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5219..5226,1..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 ctacttaggt atgtcgggaa cctctccagg gagtaccacc atgggggcag
      541 gtgccaccgg ccgcgccatg gacgggccgc gcctgctgct gttgctgctt ctgggggtgt
      601 cccttggagg tgccaaggag gcatgcccca caggcctgta cacacacagc ggtgagtgct
      661 gcaaagcctg caacctgggc gagggtgtgg cccagccttg tggagccaac cagaccgtgt
      721 gtgagccctg cctggacagc gtgacgttct ccgacgtggt gagcgcgacc gagccgtgca
      781 agccgtgcac cgagtgcgtg gggctccaga gcatgtcggc gccgtgcgtg gaggccgacg
      841 acgccgtgtg ccgctgcgcc tacggctact accaggatga gacgactggg cgctgcgagg
      901 cgtgccgcgt gtgcgaggcg ggctcgggcc tcgtgttctc ctgccaggac aagcagaaca
      961 ccgtgtgcga ggagtgcccc gacggcacgt attccgacga ggccaaccac gtggacccgt
     1021 gcctgccctg caccgtgtgc gaggacaccg agcgccagct ccgcgagtgc acacgctggg
     1081 ccgacgccga gtgcgaggag atccctggcc gttggattac acggtccaca cccccagagg
     1141 gctcggacag cacagccccc agcacccagg agcctgaggc acctccagaa caagacctca
     1201 tagccagcac ggtggcaggt gtggtgacca cagtgatggg cagctcccag cccgtggtga
     1261 cccgaggcac caccgacaac ctcatccctg tctattgctc catcctggct gctgtggttg
     1321 tgggccttgt ggcctacata gccttcaaga ggtggaacag ggggatcctc cgcggagcca
     1381 cgaacttctc tctgttaaag caagcaggag atgttgaaga aaaccccggg cccatggtgt
     1441 ccaagggcga ggaactgttc accggcgtgg tgcccatcct ggtggagctg gacggcgacg
     1501 tgaacggcca caagttcagc gtgtccggcg agggcgaagg cgacgccacc tacggcaagc
     1561 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctggtga
     1621 ccaccctgac ctacggcgtg cagtgcttca gcagataccc cgaccacatg aagcagcacg
     1681 atttcttcaa gagcgccatg cccgagggct acgtccagga gcgcaccatc tttttcaagg
     1741 acgacggcaa ctacaagacc agggccgagg tgaagttcga gggcgacaca ctggtgaaca
     1801 ggatcgagct gaagggcatc gacttcaaag aggacggcaa catcctgggc cacaagctgg
     1861 aatacaacta caacagccac aacgtgtaca ttatggccga caagcagaag aacggcatca
     1921 aggtcaactt caagatccgg cacaacatcg aggacggcag cgtgcagctg gccgaccact
     1981 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     2041 gcacccagag cgctctcagc aaggacccca acgagaagcg ggaccacatg gtgctgaaag
     2101 aatttgtgac agccgccgga atcaccctgg gcatggacga gctgtacaag agcagcggcc
     2161 ccagcggcag cagcagcctg gaagtgctgt tccagggccc cctgagcagc tctggcccct
     2221 ctggcagcgc cgccaagttc aaagaaacag ctgctgctaa attcgagcgg cagcacatgg
     2281 acagcggagg aggaggcgag aacctgtact ttcagggagg cggaggcgga ggggactata
     2341 aggacgacga cgacaagctg accgacgacc tgcccagggc ctcgggtatg tcgggaacct
     2401 ctccagggat gtctcgaggg ggggcccggt acccagcttt tgttcccttt agtgagggtt
     2461 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     2521 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     2581 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     2641 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     2701 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2761 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2821 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2881 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2941 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3001 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3061 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3121 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3181 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3241 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3301 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3361 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3421 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3481 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3541 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3601 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3661 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3721 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3781 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3841 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3901 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3961 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4021 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4081 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4141 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4201 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4261 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4321 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4381 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4441 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4501 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4561 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4621 ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4681 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     4741 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     4801 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     4861 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4921 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     4981 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     5041 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg
     5101 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     5161 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     5221 cagtca
//
