LOCUS       sequence-228075-        4183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with 3xHA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4183)
  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 4183)
  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-P28
FEATURES             Location/Qualifiers
     source          1..4183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..153
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     polyA_signal    163..284
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(200..219)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     254..273
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(291..324)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        331..647
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      498..633
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1269..1493
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1500..1748
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1500..1520
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1671..1690
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1750..1783)
                     /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(1820..1839)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1821..1839)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1926..1943)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2097..2685)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2177..2196)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2856..3647,3648..3716))
                     /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     3479..3498
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3717..3821)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3889..3907
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3945..3967)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4067..4086
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4167..4183,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4167..4183,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tcctacccat acgatgttcc tgactatgcg ggctatccct atgacgtccc ggactatgca
      121 ggatcctatc catatgacgt tccagattac gcttgaacta gtaacttgtt tattgcagct
      181 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
      241 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttagtcgat ataacttcgt
      301 atagcataca ttatacgaag ttatgtcgac ggtgtggaaa gtccccaggc tccccagcag
      361 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
      421 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
      481 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
      541 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
      601 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagaa ttcgccacca
      661 tgactgagta caagcctact gtgcgtttag ccaccaggga tgatgtgcca cgagccgtga
      721 ggacactagc agccgccttt gctgactacc cagctacaag gcacaccgtg gaccctgaca
      781 gacacattga aagagtcaca gaacttcagg agctgttttt aactagggtg ggcctggata
      841 ttggcaaagt ctgggtggcc gatgatggag cagcagttgc agtgtggacc accccagagt
      901 ctgttgaagc tggagctgtc tttgcagaaa ttggaccaag aatggccgag ttatctggca
      961 gcaggctggc tgctcaacag cagatggaag gattattagc tccccacaga cccaaggagc
     1021 ctgcctggtt cctggcaaca gttggagttt ctccagacca tcagggcaag ggcttaggat
     1081 ctgctgtggt gctgcctggt gtggaagcag ctgagagagc tggggtgcca gcatttctgg
     1141 agacatcagc tcctagaaac ctgcccttct atgaacggct gggcttcaca gtgactgctg
     1201 atgtggaggt gcctgagggc cccagaacct ggtgcatgac aagaaaacca ggagcctgac
     1261 tgcagcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     1321 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     1381 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     1441 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggctcgagg
     1501 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     1561 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     1621 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     1681 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     1741 gaaacaccga taacttcgta tagcatacat tatacgaagt tatggtaccg atgcagctag
     1801 cccgcggatc tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac
     1861 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     1921 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     1981 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2041 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2101 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2161 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2221 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2281 gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2341 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2401 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2461 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2521 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2581 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2641 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2701 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2761 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2821 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2881 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     2941 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3001 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3061 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3121 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3181 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3241 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3301 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3361 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3421 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3481 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3541 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3601 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3661 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3721 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3781 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3841 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     3901 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     3961 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     4021 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     4081 attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac
     4141 ataaccttat gtatcataca catacgattt aggtgacact ata
//
