LOCUS       sequence-228037-        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"
     CDS             64..108
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    118..239
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(155..174)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     209..228
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(246..279)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        286..534
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     286..306
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     457..476
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(536..569)
                     /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(606..625)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(607..625)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(712..729)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(883..1471)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(963..982)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1642..2433,2434..2502))
                     /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     2265..2284
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2503..2607)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2675..2693
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2731..2753)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2853..2872
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(2953..2969,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(2953..2969,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccaaagaaa ccgctgctgc taaattcgaa cgccagcaca tggacagctg aactagtaac
      121 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
      181 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttag
      241 tcgatataac ttcgtatagc atacattata cgaagttatg tcgaggaggg cctatttccc
      301 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
      361 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
      421 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
      481 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgataac
      541 ttcgtatagc atacattata cgaagttatg gtaccgatgc agctagcccg cggatctgcc
      601 ggtctcccta tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat
      661 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
      721 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
      781 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
      841 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
      901 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
      961 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1021 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg
     1081 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1141 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     1201 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     1261 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     1321 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     1381 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     1441 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     1501 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     1561 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     1621 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     1681 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     1741 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     1801 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     1861 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     1921 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     1981 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     2041 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     2101 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     2161 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     2221 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     2281 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     2341 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     2401 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     2461 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     2521 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     2581 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     2641 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     2701 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     2761 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     2821 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     2881 gcaccatatg gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat
     2941 catacacata cgatttaggt gacactata
//
