LOCUS       sequence-228082-        4117 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4117)
  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 4117)
  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-P33
FEATURES             Location/Qualifiers
     source          1..4117
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..87
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    97..218
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(134..153)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     188..207
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(225..258)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        265..581
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      432..567
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1203..1427
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1434..1682
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1434..1454
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1605..1624
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1684..1717)
                     /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(1754..1773)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1755..1773)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1860..1877)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2031..2619)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2111..2130)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2790..3581,3582..3650))
                     /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     3413..3432
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3651..3755)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3823..3841
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3879..3901)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4001..4020
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4101..4117,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4101..4117,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgattaca aggatgacga tgacaagtga actagtaact tgtttattgc agcttataat
      121 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
      181 tctagttgtg gtttgtccaa actcatcaat gtatcttagt cgatataact tcgtatagca
      241 tacattatac gaagttatgt cgacggtgtg gaaagtcccc aggctcccca gcaggcagaa
      301 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
      361 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
      421 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
      481 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
      541 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agaattcgcc accatgactg
      601 agtacaagcc tactgtgcgt ttagccacca gggatgatgt gccacgagcc gtgaggacac
      661 tagcagccgc ctttgctgac tacccagcta caaggcacac cgtggaccct gacagacaca
      721 ttgaaagagt cacagaactt caggagctgt ttttaactag ggtgggcctg gatattggca
      781 aagtctgggt ggccgatgat ggagcagcag ttgcagtgtg gaccacccca gagtctgttg
      841 aagctggagc tgtctttgca gaaattggac caagaatggc cgagttatct ggcagcaggc
      901 tggctgctca acagcagatg gaaggattat tagctcccca cagacccaag gagcctgcct
      961 ggttcctggc aacagttgga gtttctccag accatcaggg caagggctta ggatctgctg
     1021 tggtgctgcc tggtgtggaa gcagctgaga gagctggggt gccagcattt ctggagacat
     1081 cagctcctag aaacctgccc ttctatgaac ggctgggctt cacagtgact gctgatgtgg
     1141 aggtgcctga gggccccaga acctggtgca tgacaagaaa accaggagcc tgactgcagc
     1201 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     1261 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     1321 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     1381 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctc gaggagggcc
     1441 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg
     1501 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     1561 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     1621 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     1681 ccgataactt cgtatagcat acattatacg aagttatggt accgatgcag ctagcccgcg
     1741 gatctgccgg tctccctata gtgagtcgta ttaatttcga taagccaggt taacctgcat
     1801 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     1861 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     1921 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     1981 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2041 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2101 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2161 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2221 tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     2281 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     2341 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     2401 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     2461 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     2521 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2581 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     2641 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     2701 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     2761 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     2821 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     2881 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     2941 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3001 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3061 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3121 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3181 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3241 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     3301 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     3361 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     3421 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3481 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     3541 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     3601 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     3661 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     3721 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     3781 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     3841 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     3901 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     3961 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
     4021 ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc
     4081 ttatgtatca tacacatacg atttaggtga cactata
//
