LOCUS       sequence-228036-        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"
     CDS             64..105
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     polyA_signal    115..236
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(152..171)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     206..225
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(243..276)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        283..531
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     283..303
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     454..473
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(533..566)
                     /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(603..622)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(604..622)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(709..726)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(880..1468)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(960..979)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1639..2430,2431..2499))
                     /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     2262..2281
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2500..2604)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2672..2690
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2728..2750)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2850..2869
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(2950..2966,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(2950..2966,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgtgaac tagtaacttg
      121 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
      181 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttagtcg
      241 atataacttc gtatagcata cattatacga agttatgtcg aggagggcct atttcccatg
      301 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
      361 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
      421 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
      481 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgataacttc
      541 gtatagcata cattatacga agttatggta ccgatgcagc tagcccgcgg atctgccggt
      601 ctccctatag tgagtcgtat taatttcgat aagccaggtt aacctgcatt aatgaatcgg
      661 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
      721 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
      781 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
      841 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
      901 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
      961 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     1021 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc
     1081 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     1141 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     1201 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     1261 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     1321 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     1381 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     1441 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     1501 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     1561 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     1621 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     1681 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     1741 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     1801 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     1861 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     1921 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     1981 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     2041 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     2101 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     2161 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     2221 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     2281 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     2341 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     2401 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     2461 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     2521 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     2581 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     2641 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
     2701 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     2761 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     2821 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     2881 ccatatggac atattgtcgt tagaacgcgg ctacaattaa tacataacct tatgtatcat
     2941 acacatacga tttaggtgac actata
//
