LOCUS       sequence-228071-        4636 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with TAP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4636)
  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 4636)
  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-P24
FEATURES             Location/Qualifiers
     source          1..4636
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..141
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     CDS             169..189
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             229..402
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             403..576
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNGAQAPK"
     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..1100
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      951..1086
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1722..1946
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1953..2201
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1953..1973
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2124..2143
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2203..2236)
                     /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(2273..2292)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2274..2292)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2379..2396)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2550..3138)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2630..2649)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3309..4100,4101..4169))
                     /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     3932..3951
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4170..4274)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4342..4360
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4398..4420)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4520..4539
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4620..4636,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4620..4636,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccaagagaa gatggaaaaa gaatttcata gccgtctcag cagccaaccg ctttaagaaa
      121 atctcatcct ccggggcact tgattatgat attccaacta ctgctagcga gaatttgtat
      181 tttcagggtg agctcaaaac cgcggctctt gcgcaacacg atgaagccgt ggacaacaaa
      241 ttcaacaaag aacaacaaaa cgcgttctat gagatcttac atttacctaa cttaaacgaa
      301 gaacaacgaa acgccttcat ccaaagttta aaagatgacc caagccaaag cgctaacctt
      361 ttagcagaag ctaaaaagct aaatgatgct caggcgccga aagtagacaa caaattcaac
      421 aaagaacaac aaaacgcgtt ctatgagatc ttacatttac ctaacttaaa cgaagaacaa
      481 cgaaacgcct tcatccaaag tttaaaagat gacccaagcc aaagcgctaa ccttttagca
      541 gaagctaaaa agctaaatgg tgctcaggcg ccgaaagtag acgcgaattc cgcggggaag
      601 tcaacctgaa ctagtaactt gtttattgca gcttataatg gttacaaata aagcaatagc
      661 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
      721 ctcatcaatg tatcttagtc gatataactt cgtatagcat acattatacg aagttatgtc
      781 gacggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
      841 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
      901 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
      961 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     1021 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     1081 gaggcctagg cttttgcaaa gaattcgcca ccatgactga gtacaagcct actgtgcgtt
     1141 tagccaccag ggatgatgtg ccacgagccg tgaggacact agcagccgcc tttgctgact
     1201 acccagctac aaggcacacc gtggaccctg acagacacat tgaaagagtc acagaacttc
     1261 aggagctgtt tttaactagg gtgggcctgg atattggcaa agtctgggtg gccgatgatg
     1321 gagcagcagt tgcagtgtgg accaccccag agtctgttga agctggagct gtctttgcag
     1381 aaattggacc aagaatggcc gagttatctg gcagcaggct ggctgctcaa cagcagatgg
     1441 aaggattatt agctccccac agacccaagg agcctgcctg gttcctggca acagttggag
     1501 tttctccaga ccatcagggc aagggcttag gatctgctgt ggtgctgcct ggtgtggaag
     1561 cagctgagag agctggggtg ccagcatttc tggagacatc agctcctaga aacctgccct
     1621 tctatgaacg gctgggcttc acagtgactg ctgatgtgga ggtgcctgag ggccccagaa
     1681 cctggtgcat gacaagaaaa ccaggagcct gactgcagcg actgtgcctt ctagttgcca
     1741 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     1801 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     1861 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     1921 tgctggggat gcggtgggct ctatggctcg aggagggcct atttcccatg attccttcat
     1981 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     2041 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     2101 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     2161 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgataacttc gtatagcata
     2221 cattatacga agttatggta ccgatgcagc tagcccgcgg atctgccggt ctccctatag
     2281 tgagtcgtat taatttcgat aagccaggtt aacctgcatt aatgaatcgg ccaacgcgcg
     2341 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     2401 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     2461 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     2521 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2581 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2641 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2701 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg
     2761 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2821 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2881 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2941 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     3001 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3061 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3121 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3181 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     3241 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3301 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3361 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3421 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3481 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3541 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3601 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3661 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3721 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3781 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3841 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3901 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3961 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4021 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4081 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4141 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4201 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4261 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     4321 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     4381 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     4441 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     4501 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatggac
     4561 atattgtcgt tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga
     4621 tttaggtgac actata
//
