LOCUS       sequence-232685-        3910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with V5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3910)
  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 3910)
  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-Z31
FEATURES             Location/Qualifiers
     source          1..3910
                     /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"
     regulatory      623..632
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             629..1003
                     /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    1013..1237
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1244..1492
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1244..1264
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1415..1434
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1494..1527)
                     /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(1564..1583)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1565..1583)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1670..1687)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1841..2429)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1921..1940)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2600..3391,3392..3460))
                     /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     3223..3242
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3461..3565)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3633..3651
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3689..3711)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3811..3830
                     /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 ggccaagttg accagtgccg ttccggtgct
      661 caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg
      721 ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag
      781 cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct
      841 ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg
      901 gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc
      961 ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgactgcagc gactgtgcct
     1021 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     1081 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     1141 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     1201 aatagcaggc atgctgggga tgcggtgggc tctatggctc gaggagggcc tatttcccat
     1261 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
     1321 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     1381 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     1441 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgataactt
     1501 cgtatagcat acattatacg aagttatggt accgatgcag ctagcccgcg gatctgccgg
     1561 tctccctata gtgagtcgta ttaatttcga taagccaggt taacctgcat taatgaatcg
     1621 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     1681 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     1741 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     1801 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     1861 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     1921 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     1981 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2041 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2101 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     2161 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     2221 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     2281 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     2341 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     2401 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     2461 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     2521 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     2581 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     2641 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     2701 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     2761 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     2821 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     2881 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     2941 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3001 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3061 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     3121 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     3181 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     3241 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     3301 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     3361 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     3421 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     3481 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     3541 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     3601 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     3661 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     3721 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     3781 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     3841 accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc ttatgtatca
     3901 tacacatacg
//
