LOCUS       sequence-232608-        2969 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with S-Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2969)
  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 2969)
  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-32
FEATURES             Location/Qualifiers
     source          1..2969
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             81..125
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    135..256
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(172..191)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     226..245
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(263..296)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        303..551
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     303..323
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     474..493
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(553..586)
                     /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(623..642)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(624..642)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(729..746)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(900..1488)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(980..999)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1659..2450,2451..2519))
                     /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     2282..2301
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2520..2624)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2692..2710
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2748..2770)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2870..2889
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc aaagaaaccg ctgctgctaa attcgaacgc cagcacatgg
      121 acagctgaac tagtaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
      181 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
      241 tcatcaatgt atcttagtcg atataacttc gtatagcata cattatacga agttatgtcg
      301 aggagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
      361 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
      421 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
      481 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
      541 gacgaaacac cgataacttc gtatagcata cattatacga agttatggta ccgatgcagc
      601 tagcccgcgg atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt
      661 aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
      721 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
      781 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
      841 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
      901 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
      961 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     1021 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     1081 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     1141 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     1201 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     1261 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     1321 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     1381 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     1441 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     1501 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     1561 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     1621 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     1681 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     1741 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     1801 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     1861 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     1921 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     1981 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     2041 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     2101 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     2161 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     2221 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     2281 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     2341 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     2401 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     2461 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     2521 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     2581 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     2641 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     2701 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     2761 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     2821 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     2881 cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa
     2941 tacataacct tatgtatcat acacatacg
//
