LOCUS       sequence-384036-        5409 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 5409)
  AUTHORS   Thone FMB, Kurrle NS, von Melchner H, Schnutgen F
  TITLE     CRISPR/Cas9-mediated generic protein tagging in mammalian cells.
  JOURNAL   Methods. 2019 Jul 15;164-165:59-66. doi: 
            10.1016/j.ymeth.2019.02.018. Epub 2019 Feb 22.
  PUBMED    30797903
REFERENCE   2  (bases 1 to 5409)
  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: "Methods."; 
            date: "2019-07-15"; volume: "164-165:59-66. doi"; pages: " 
            10.1016/j.ymeth.2019.02.018. Epub 2019 Feb 22"
COMMENT     Sequence Label: pGTag Hygro EGFP nLAP +2
FEATURES             Location/Qualifiers
     source          1..5409
                     /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             531..1553
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             531..1553
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1560..1616
                     /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(1923..1942)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2361..2384
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2424..2468
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             2481..2501
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2517..2540
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2629..2649)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2629..2647)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2668..2684)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2668..2684)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2681..2703)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2692..2708
                     /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(2716..2722,2723..2740,2741..2746))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2761..2782
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2899..2916)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3070..3658)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3150..3169)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3829..4620,4621..4689))
                     /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     4452..4471
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4690..4794)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4821..5276
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4908..4927)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5118..5139
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5402..5409,1..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 ataccgtcat ctacttaggt atgtcgggaa cctctccagg gagtaccacc atgaaaaagc
      541 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
      601 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
      661 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
      721 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
      781 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
      841 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
      901 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
      961 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
     1021 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
     1081 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
     1141 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
     1201 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
     1261 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
     1321 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     1381 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     1441 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     1501 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaacgcggag
     1561 ccacgaactt ctctctgtta aagcaagcag gagatgttga agaaaacccc gggcccatgg
     1621 tgtccaaggg cgaggaactg ttcaccggcg tggtgcccat cctggtggag ctggacggcg
     1681 acgtgaacgg ccacaagttc agcgtgtccg gcgagggcga aggcgacgcc acctacggca
     1741 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctgg
     1801 tgaccaccct gacctacggc gtgcagtgct tcagcagata ccccgaccac atgaagcagc
     1861 acgatttctt caagagcgcc atgcccgagg gctacgtcca ggagcgcacc atctttttca
     1921 aggacgacgg caactacaag accagggccg aggtgaagtt cgagggcgac acactggtga
     1981 acaggatcga gctgaagggc atcgacttca aagaggacgg caacatcctg ggccacaagc
     2041 tggaatacaa ctacaacagc cacaacgtgt acattatggc cgacaagcag aagaacggca
     2101 tcaaggtcaa cttcaagatc cggcacaaca tcgaggacgg cagcgtgcag ctggccgacc
     2161 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2221 tgagcaccca gagcgctctc agcaaggacc ccaacgagaa gcgggaccac atggtgctga
     2281 aagaatttgt gacagccgcc ggaatcaccc tgggcatgga cgagctgtac aagagcagcg
     2341 gccccagcgg cagcagcagc ctggaagtgc tgttccaggg ccccctgagc agctctggcc
     2401 cctctggcag cgccgccaag ttcaaagaaa cagctgctgc taaattcgag cggcagcaca
     2461 tggacagcgg aggaggaggc gagaacctgt actttcaggg aggcggaggc ggaggggact
     2521 ataaggacga cgacgacaag ctgaccgacg acctgcccag ggcctcgggt atgtcgggaa
     2581 cctctccagg gatgtctcga gggggggccc ggtacccagc ttttgttccc tttagtgagg
     2641 gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     2701 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     2761 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2821 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     2881 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     2941 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3001 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3061 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3121 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3181 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3241 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3301 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3361 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3421 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3481 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3541 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3601 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3661 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3721 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3781 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3841 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     3901 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     3961 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4021 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4081 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4141 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4201 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     4261 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     4321 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4381 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4441 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4501 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4561 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4621 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4681 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4741 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4801 tccccgaaaa gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4861 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     4921 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4981 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     5041 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     5101 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     5161 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     5221 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttccat
     5281 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     5341 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     5401 tcccagtca
//
