LOCUS       sequence-228081-        4138 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with S-Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4138)
  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 4138)
  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-P32
FEATURES             Location/Qualifiers
     source          1..4138
                     /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..602
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      453..588
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1224..1448
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1455..1703
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1455..1475
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1626..1645
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1705..1738)
                     /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(1775..1794)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1776..1794)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1881..1898)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2052..2640)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2132..2151)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2811..3602,3603..3671))
                     /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     3434..3453
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3672..3776)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3844..3862
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3900..3922)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4022..4041
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4122..4138,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4122..4138,1)
                     /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 tcgacggtgt ggaaagtccc
      301 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
      361 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
      421 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
      481 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
      541 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
      601 aagaattcgc caccatgact gagtacaagc ctactgtgcg tttagccacc agggatgatg
      661 tgccacgagc cgtgaggaca ctagcagccg cctttgctga ctacccagct acaaggcaca
      721 ccgtggaccc tgacagacac attgaaagag tcacagaact tcaggagctg tttttaacta
      781 gggtgggcct ggatattggc aaagtctggg tggccgatga tggagcagca gttgcagtgt
      841 ggaccacccc agagtctgtt gaagctggag ctgtctttgc agaaattgga ccaagaatgg
      901 ccgagttatc tggcagcagg ctggctgctc aacagcagat ggaaggatta ttagctcccc
      961 acagacccaa ggagcctgcc tggttcctgg caacagttgg agtttctcca gaccatcagg
     1021 gcaagggctt aggatctgct gtggtgctgc ctggtgtgga agcagctgag agagctgggg
     1081 tgccagcatt tctggagaca tcagctccta gaaacctgcc cttctatgaa cggctgggct
     1141 tcacagtgac tgctgatgtg gaggtgcctg agggccccag aacctggtgc atgacaagaa
     1201 aaccaggagc ctgactgcag cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     1261 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     1321 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     1381 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     1441 ctctatggct cgaggagggc ctatttccca tgattccttc atatttgcat atacgataca
     1501 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     1561 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     1621 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     1681 tcttgtggaa aggacgaaac accgataact tcgtatagca tacattatac gaagttatgg
     1741 taccgatgca gctagcccgc ggatctgccg gtctccctat agtgagtcgt attaatttcg
     1801 ataagccagg ttaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     1861 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     1921 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     1981 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     2041 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2101 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     2161 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     2221 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag
     2281 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     2341 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     2401 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     2461 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     2521 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     2581 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     2641 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2701 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2761 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     2821 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2881 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     2941 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     3001 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3061 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3121 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3181 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3241 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3301 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3361 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3421 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     3481 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     3541 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     3601 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     3661 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     3721 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     3781 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     3841 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     3901 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     3961 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     4021 gcggcatcag agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc
     4081 ggctacaatt aatacataac cttatgtatc atacacatac gatttaggtg acactata
//
