LOCUS       sequence-232606-        2957 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with VSV-G.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2957)
  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 2957)
  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-30
FEATURES             Location/Qualifiers
     source          1..2957
                     /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..113
                     /codon_start=1
                     /product="epitope tag from vesicular stomatitis virus G 
                     protein"
                     /label=VSV-G tag
                     /translation="YTDIEMNRLGK"
     polyA_signal    123..244
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(160..179)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     214..233
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(251..284)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        291..539
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     291..311
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     462..481
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(541..574)
                     /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(611..630)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(612..630)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(717..734)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(888..1476)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(968..987)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1647..2438,2439..2507))
                     /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     2270..2289
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2508..2612)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2680..2698
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2736..2758)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2858..2877
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc tatacagaca tagagatgaa ccgacttgga aagtgaacta
      121 gtaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
      181 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
      241 cttagtcgat ataacttcgt atagcataca ttatacgaag ttatgtcgag gagggcctat
      301 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
      361 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
      421 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
      481 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
      541 ataacttcgt atagcataca ttatacgaag ttatggtacc gatgcagcta gcccgcggat
      601 ctgccggtct ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa
      661 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
      721 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
      781 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
      841 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
      901 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      961 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     1021 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     1081 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     1141 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1201 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     1261 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     1321 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     1381 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     1441 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     1501 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     1561 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     1621 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     1681 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     1741 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     1801 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     1861 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     1921 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     1981 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     2041 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     2101 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     2161 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     2221 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     2281 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     2341 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     2401 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     2461 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     2521 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     2581 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     2641 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     2701 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     2761 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     2821 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     2881 agagtgcacc atatggacat attgtcgtta gaacgcggct acaattaata cataacctta
     2941 tgtatcatac acatacg
//
