LOCUS       sequence-232676-        4411 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with TAP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4411)
  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 4411)
  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-Z24
FEATURES             Location/Qualifiers
     source          1..4411
                     /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"
     regulatory      1124..1133
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1130..1504
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    1514..1738
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1745..1993
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1745..1765
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1916..1935
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1995..2028)
                     /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(2065..2084)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2066..2084)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2171..2188)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2342..2930)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2422..2441)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3101..3892,3893..3961))
                     /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     3724..3743
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3962..4066)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4134..4152
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4190..4212)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4312..4331
                     /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 tggccaagtt
     1141 gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac
     1201 cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga
     1261 cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc
     1321 ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac
     1381 gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg
     1441 ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga
     1501 ctgactgcag cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     1561 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     1621 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     1681 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     1741 cgaggagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     1801 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     1861 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     1921 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     1981 aggacgaaac accgataact tcgtatagca tacattatac gaagttatgg taccgatgca
     2041 gctagcccgc ggatctgccg gtctccctat agtgagtcgt attaatttcg ataagccagg
     2101 ttaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2161 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2221 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2281 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2341 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2401 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2461 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2521 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2581 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2641 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2701 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2761 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     2821 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     2881 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     2941 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3001 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3061 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3121 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3181 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3241 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3301 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3361 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3421 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3481 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3541 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3601 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3661 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3721 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3781 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     3841 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     3901 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     3961 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4021 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4081 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     4141 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     4201 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     4261 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     4321 agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt
     4381 aatacataac cttatgtatc atacacatac g
//
