LOCUS       sequence-232649-        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"
     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..619
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      470..605
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1241..1465
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1472..1720
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1472..1492
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1643..1662
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1722..1755)
                     /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(1792..1811)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1793..1811)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1898..1915)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2069..2657)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2149..2168)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2828..3619,3620..3688))
                     /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     3451..3470
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3689..3793)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3861..3879
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3917..3939)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4039..4058
                     /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 acggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
      361 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
      421 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
      481 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
      541 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
      601 aggcctaggc ttttgcaaag aattcgccac catgactgag tacaagccta ctgtgcgttt
      661 agccaccagg gatgatgtgc cacgagccgt gaggacacta gcagccgcct ttgctgacta
      721 cccagctaca aggcacaccg tggaccctga cagacacatt gaaagagtca cagaacttca
      781 ggagctgttt ttaactaggg tgggcctgga tattggcaaa gtctgggtgg ccgatgatgg
      841 agcagcagtt gcagtgtgga ccaccccaga gtctgttgaa gctggagctg tctttgcaga
      901 aattggacca agaatggccg agttatctgg cagcaggctg gctgctcaac agcagatgga
      961 aggattatta gctccccaca gacccaagga gcctgcctgg ttcctggcaa cagttggagt
     1021 ttctccagac catcagggca agggcttagg atctgctgtg gtgctgcctg gtgtggaagc
     1081 agctgagaga gctggggtgc cagcatttct ggagacatca gctcctagaa acctgccctt
     1141 ctatgaacgg ctgggcttca cagtgactgc tgatgtggag gtgcctgagg gccccagaac
     1201 ctggtgcatg acaagaaaac caggagcctg actgcagcga ctgtgccttc tagttgccag
     1261 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     1321 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     1381 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     1441 gctggggatg cggtgggctc tatggctcga ggagggccta tttcccatga ttccttcata
     1501 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     1561 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     1621 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     1681 tttcttggct ttatatatct tgtggaaagg acgaaacacc gataacttcg tatagcatac
     1741 attatacgaa gttatggtac cgatgcagct agcccgcgga tctgccggtc tccctatagt
     1801 gagtcgtatt aatttcgata agccaggtta acctgcatta atgaatcggc caacgcgcgg
     1861 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     1921 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     1981 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     2041 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2101 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2161 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2221 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     2281 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2341 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     2401 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2461 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     2521 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2581 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2641 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2701 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2761 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2821 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2881 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2941 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3001 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3061 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3121 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3181 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3241 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3301 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3361 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3421 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3481 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3541 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3601 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3661 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     3721 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3781 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     3841 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     3901 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     3961 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     4021 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatggaca
     4081 tattgtcgtt agaacgcggc tacaattaat acataacctt atgtatcata cacatacg
//
