LOCUS       sequence-389109-        5229 bp    DNA     circular SYN 15-APR-2025
DEFINITION  CRISPR/Cas9-mediated generic protein tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5229)
  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 5229)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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..5229
                     /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(2449..2469)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2449..2467)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2488..2504)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2488..2504)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2501..2523)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2512..2528
                     /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(2536..2542,2543..2560,2561..2566))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2581..2602
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2719..2736)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2890..3478)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2970..2989)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3649..4440,4441..4509))
                     /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     4272..4291
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4510..4614)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4641..5096
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4728..4747)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4938..4959
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5222..5229,1..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 ctactccctg gagaggttcc cgacatacct aagtaccacc 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 ctcgttcctg gagaggttcc
     2401 cgacatacct gatgtctcga gggggggccc ggtacccagc ttttgttccc tttagtgagg
     2461 gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     2521 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     2581 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2641 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     2701 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     2761 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     2821 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2881 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2941 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3001 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3061 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3121 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3181 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3241 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3301 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3361 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3421 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3481 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3541 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3601 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3661 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     3721 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     3781 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     3841 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     3901 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     3961 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4021 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     4081 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     4141 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4201 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4261 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4321 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4381 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4441 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4501 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4561 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4621 tccccgaaaa gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4681 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     4741 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4801 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     4861 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     4921 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     4981 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     5041 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttccat
     5101 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     5161 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     5221 tcccagtca
//
