LOCUS       sequence-232648-        4135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with V5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4135)
  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 4135)
  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-P31
FEATURES             Location/Qualifiers
     source          1..4135
                     /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..616
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      467..602
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1238..1462
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1469..1717
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1469..1489
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1640..1659
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1719..1752)
                     /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(1789..1808)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1790..1808)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1895..1912)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2066..2654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2146..2165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2825..3616,3617..3685))
                     /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     3448..3467
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3686..3790)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3858..3876
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3914..3936)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4036..4055
                     /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 tatgtcgacg
      301 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
      361 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
      421 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
      481 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
      541 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
      601 cctaggcttt tgcaaagaat tcgccaccat gactgagtac aagcctactg tgcgtttagc
      661 caccagggat gatgtgccac gagccgtgag gacactagca gccgcctttg ctgactaccc
      721 agctacaagg cacaccgtgg accctgacag acacattgaa agagtcacag aacttcagga
      781 gctgttttta actagggtgg gcctggatat tggcaaagtc tgggtggccg atgatggagc
      841 agcagttgca gtgtggacca ccccagagtc tgttgaagct ggagctgtct ttgcagaaat
      901 tggaccaaga atggccgagt tatctggcag caggctggct gctcaacagc agatggaagg
      961 attattagct ccccacagac ccaaggagcc tgcctggttc ctggcaacag ttggagtttc
     1021 tccagaccat cagggcaagg gcttaggatc tgctgtggtg ctgcctggtg tggaagcagc
     1081 tgagagagct ggggtgccag catttctgga gacatcagct cctagaaacc tgcccttcta
     1141 tgaacggctg ggcttcacag tgactgctga tgtggaggtg cctgagggcc ccagaacctg
     1201 gtgcatgaca agaaaaccag gagcctgact gcagcgactg tgccttctag ttgccagcca
     1261 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     1321 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     1381 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     1441 ggggatgcgg tgggctctat ggctcgagga gggcctattt cccatgattc cttcatattt
     1501 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
     1561 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     1621 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     1681 cttggcttta tatatcttgt ggaaaggacg aaacaccgat aacttcgtat agcatacatt
     1741 atacgaagtt atggtaccga tgcagctagc ccgcggatct gccggtctcc ctatagtgag
     1801 tcgtattaat ttcgataagc caggttaacc tgcattaatg aatcggccaa cgcgcgggga
     1861 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     1921 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     1981 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2041 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2101 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2161 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2221 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2281 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2341 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2401 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2461 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2521 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2581 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2641 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2701 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2761 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2821 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2881 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2941 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3001 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3061 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3121 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3181 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3241 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3301 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3361 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3421 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3481 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3541 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3601 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3661 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3721 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3781 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     3841 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     3901 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     3961 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     4021 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atggacatat
     4081 tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac atacg
//
