LOCUS       sequence-232641-        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"
     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..158
                     /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             186..206
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             246..419
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             420..593
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNGAQAPK"
     polyA_signal    633..754
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(670..689)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     724..743
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(761..794)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        801..1117
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      968..1103
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1739..1963
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1970..2218
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1970..1990
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2141..2160
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2220..2253)
                     /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(2290..2309)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2291..2309)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2396..2413)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2567..3155)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2647..2666)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3326..4117,4118..4186))
                     /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     3949..3968
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4187..4291)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4359..4377
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4415..4437)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4537..4556
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc aagagaagat ggaaaaagaa tttcatagcc gtctcagcag
      121 ccaaccgctt taagaaaatc tcatcctccg gggcacttga ttatgatatt ccaactactg
      181 ctagcgagaa tttgtatttt cagggtgagc tcaaaaccgc ggctcttgcg caacacgatg
      241 aagccgtgga caacaaattc aacaaagaac aacaaaacgc gttctatgag atcttacatt
      301 tacctaactt aaacgaagaa caacgaaacg ccttcatcca aagtttaaaa gatgacccaa
      361 gccaaagcgc taacctttta gcagaagcta aaaagctaaa tgatgctcag gcgccgaaag
      421 tagacaacaa attcaacaaa gaacaacaaa acgcgttcta tgagatctta catttaccta
      481 acttaaacga agaacaacga aacgccttca tccaaagttt aaaagatgac ccaagccaaa
      541 gcgctaacct tttagcagaa gctaaaaagc taaatggtgc tcaggcgccg aaagtagacg
      601 cgaattccgc ggggaagtca acctgaacta gtaacttgtt tattgcagct tataatggtt
      661 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
      721 gttgtggttt gtccaaactc atcaatgtat cttagtcgat ataacttcgt atagcataca
      781 ttatacgaag ttatgtcgac ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
      841 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
      901 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
      961 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     1021 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     1081 gtgaggaggc ttttttggag gcctaggctt ttgcaaagaa ttcgccacca tgactgagta
     1141 caagcctact gtgcgtttag ccaccaggga tgatgtgcca cgagccgtga ggacactagc
     1201 agccgccttt gctgactacc cagctacaag gcacaccgtg gaccctgaca gacacattga
     1261 aagagtcaca gaacttcagg agctgttttt aactagggtg ggcctggata ttggcaaagt
     1321 ctgggtggcc gatgatggag cagcagttgc agtgtggacc accccagagt ctgttgaagc
     1381 tggagctgtc tttgcagaaa ttggaccaag aatggccgag ttatctggca gcaggctggc
     1441 tgctcaacag cagatggaag gattattagc tccccacaga cccaaggagc ctgcctggtt
     1501 cctggcaaca gttggagttt ctccagacca tcagggcaag ggcttaggat ctgctgtggt
     1561 gctgcctggt gtggaagcag ctgagagagc tggggtgcca gcatttctgg agacatcagc
     1621 tcctagaaac ctgcccttct atgaacggct gggcttcaca gtgactgctg atgtggaggt
     1681 gcctgagggc cccagaacct ggtgcatgac aagaaaacca ggagcctgac tgcagcgact
     1741 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     1801 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     1861 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     1921 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggctcgagg agggcctatt
     1981 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
     2041 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     2101 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     2161 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga
     2221 taacttcgta tagcatacat tatacgaagt tatggtaccg atgcagctag cccgcggatc
     2281 tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac ctgcattaat
     2341 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     2401 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     2461 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     2521 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     2581 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     2641 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2701 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     2761 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     2821 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     2881 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     2941 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3001 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3061 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3121 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3181 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3241 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3301 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3361 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3421 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3481 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3541 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3601 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3661 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3721 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     3781 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     3841 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     3901 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     3961 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4021 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4081 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4141 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4201 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4261 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     4321 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     4381 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     4441 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     4501 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
     4561 gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac ataaccttat
     4621 gtatcataca catacg
//
