LOCUS       sequence-232607-        2966 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with V5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2966)
  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 2966)
  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-31
FEATURES             Location/Qualifiers
     source          1..2966
                     /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..122
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     polyA_signal    132..253
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(169..188)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     223..242
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(260..293)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        300..548
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     300..320
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     471..490
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(550..583)
                     /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(620..639)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(621..639)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(726..743)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(897..1485)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(977..996)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1656..2447,2448..2516))
                     /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     2279..2298
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2517..2621)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2689..2707
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2745..2767)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2867..2886
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc ggtaagccta tccctaaccc tctcctcggt ctcgattcta
      121 cgtgaactag taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
      181 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
      241 tcaatgtatc ttagtcgata taacttcgta tagcatacat tatacgaagt tatgtcgagg
      301 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
      361 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
      421 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
      481 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
      541 gaaacaccga taacttcgta tagcatacat tatacgaagt tatggtaccg atgcagctag
      601 cccgcggatc tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac
      661 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
      721 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
      781 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
      841 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
      901 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
      961 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     1021 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     1081 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     1141 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     1201 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     1261 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     1321 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     1381 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     1441 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     1501 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     1561 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     1621 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     1681 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     1741 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     1801 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     1861 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     1921 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     1981 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     2041 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     2101 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     2161 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     2221 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     2281 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     2341 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     2401 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     2461 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     2521 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     2581 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     2641 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     2701 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     2761 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     2821 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     2881 attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac
     2941 ataaccttat gtatcataca catacg
//
