LOCUS       sequence-232644-        4120 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with 1xHA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4120)
  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 4120)
  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-P27
FEATURES             Location/Qualifiers
     source          1..4120
                     /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..107
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    117..238
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(154..173)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     208..227
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(245..278)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        285..601
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      452..587
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1223..1447
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1454..1702
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1454..1474
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1625..1644
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1704..1737)
                     /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(1774..1793)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1775..1793)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1880..1897)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2051..2639)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2131..2150)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2810..3601,3602..3670))
                     /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     3433..3452
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3671..3775)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3843..3861
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3899..3921)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4021..4040
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc tacccatacg acgtcccaga ctacgcttga actagtaact
      121 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
      181 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttagt
      241 cgatataact tcgtatagca tacattatac gaagttatgt cgacggtgtg gaaagtcccc
      301 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
      361 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
      421 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
      481 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
      541 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
      601 agaattcgcc accatgactg agtacaagcc tactgtgcgt ttagccacca gggatgatgt
      661 gccacgagcc gtgaggacac tagcagccgc ctttgctgac tacccagcta caaggcacac
      721 cgtggaccct gacagacaca ttgaaagagt cacagaactt caggagctgt ttttaactag
      781 ggtgggcctg gatattggca aagtctgggt ggccgatgat ggagcagcag ttgcagtgtg
      841 gaccacccca gagtctgttg aagctggagc tgtctttgca gaaattggac caagaatggc
      901 cgagttatct ggcagcaggc tggctgctca acagcagatg gaaggattat tagctcccca
      961 cagacccaag gagcctgcct ggttcctggc aacagttgga gtttctccag accatcaggg
     1021 caagggctta ggatctgctg tggtgctgcc tggtgtggaa gcagctgaga gagctggggt
     1081 gccagcattt ctggagacat cagctcctag aaacctgccc ttctatgaac ggctgggctt
     1141 cacagtgact gctgatgtgg aggtgcctga gggccccaga acctggtgca tgacaagaaa
     1201 accaggagcc tgactgcagc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     1261 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     1321 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     1381 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     1441 tctatggctc gaggagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     1501 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     1561 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     1621 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     1681 cttgtggaaa ggacgaaaca ccgataactt cgtatagcat acattatacg aagttatggt
     1741 accgatgcag ctagcccgcg gatctgccgg tctccctata gtgagtcgta ttaatttcga
     1801 taagccaggt taacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     1861 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     1921 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     1981 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2041 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2101 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2161 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     2221 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     2281 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     2341 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     2401 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     2461 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     2521 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     2581 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     2641 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     2701 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     2761 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     2821 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     2881 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     2941 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3001 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3061 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3121 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3181 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     3241 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     3301 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     3361 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     3421 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     3481 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     3541 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     3601 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     3661 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     3721 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     3781 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     3841 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc
     3901 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     3961 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
     4021 cggcatcaga gcagattgta ctgagagtgc accatatgga catattgtcg ttagaacgcg
     4081 gctacaatta atacataacc ttatgtatca tacacatacg
//
