LOCUS       sequence-228027-        3467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with SNAP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3467)
  AUTHORS   Fueller J, Herbst K, Meurer M, Gubicza K, Kurtulmus B, Knopf JD, 
            Kirrmaier D, Buchmuller BC, Pereira G, Lemberg MK, Knop M
  TITLE     CRISPR-Cas12a-assisted PCR tagging of mammalian genes.
  JOURNAL   J Cell Biol. 2020 Jun 1;219(6). pii: 151766. doi: 
            10.1083/jcb.201910210.
  PUBMED    32406907
REFERENCE   2  (bases 1 to 3467)
  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: "J Cell 
            Biol."; date: "2020-06-1"; volume: "219(6). pii: 151766. doi"; 
            pages: " 10.1083/jcb.201910210"
COMMENT     Sequence Label: pMaCTag-22
FEATURES             Location/Qualifiers
     source          1..3467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..606
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for mammalian expression"
                     /translation="DKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVEV
                     PAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKV
                     VKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLAV
                     KEWLLAHEGHRLGKPGLG"
     polyA_signal    616..737
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(653..672)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     707..726
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(744..777)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        784..1032
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     784..804
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     955..974
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1034..1067)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1104..1123)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1105..1123)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1210..1227)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1381..1969)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1461..1480)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2140..2931,2932..3000))
                     /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     2763..2782
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3001..3105)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3173..3191
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3229..3251)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3351..3370
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3451..3467,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3451..3467,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgacaaag actgcgaaat gaagcgcacc accctggata gccctctggg caagctggaa
      121 ctgtctgggt gcgaacaggg cctgcacgag atcaagctgc tgggcaaagg aacatctgcc
      181 gccgacgccg tggaagtgcc tgccccagcc gccgtgctgg gcggaccaga gccactgatg
      241 caggccaccg cctggctcaa cgcctacttt caccagcctg aggccatcga ggagttccct
      301 gtgccagccc tgcaccaccc agtgttccag caggagagct ttacccgcca ggtgctgtgg
      361 aaactgctga aagtggtgaa gttcggagag gtcatcagct accagcagct ggccgccctg
      421 gccggcaatc ccgccgccac cgccgccgtg aaaaccgccc tgagcggaaa tcccgtgccc
      481 attctgatcc cctgccaccg ggtggtgtct agctctggcg ccgtgggggg ctacgagggc
      541 gggctcgccg tgaaagagtg gctgctggcc cacgagggcc acagactggg caagcctggg
      601 ctgggttgaa ctagtaactt gtttattgca gcttataatg gttacaaata aagcaatagc
      661 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
      721 ctcatcaatg tatcttagtc gatataactt cgtatagcat acattatacg aagttatgtc
      781 gaggagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
      841 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
      901 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
      961 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     1021 ggacgaaaca ccgataactt cgtatagcat acattatacg aagttatggt accgatgcag
     1081 ctagcccgcg gatctgccgg tctccctata gtgagtcgta ttaatttcga taagccaggt
     1141 taacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     1201 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     1261 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     1321 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     1381 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     1441 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     1501 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     1561 cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     1621 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     1681 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     1741 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     1801 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     1861 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     1921 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     1981 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     2041 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     2101 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     2161 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     2221 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     2281 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     2341 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     2401 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     2461 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     2521 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     2581 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     2641 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     2701 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     2761 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     2821 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     2881 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     2941 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     3001 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     3061 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     3121 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     3181 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     3241 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     3301 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     3361 gcagattgta ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta
     3421 atacataacc ttatgtatca tacacatacg atttaggtga cactata
//
