LOCUS       sequence-232604-        3014 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with 3xHA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3014)
  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 3014)
  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-28
FEATURES             Location/Qualifiers
     source          1..3014
                     /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..170
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     polyA_signal    180..301
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(217..236)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     271..290
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(308..341)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        348..596
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     348..368
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     519..538
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(598..631)
                     /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(668..687)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(669..687)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(774..791)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(945..1533)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1025..1044)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1704..2495,2496..2564))
                     /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     2327..2346
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2565..2669)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2737..2755
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2793..2815)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2915..2934
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc tacccatacg atgttcctga ctatgcgggc tatccctatg
      121 acgtcccgga ctatgcagga tcctatccat atgacgttcc agattacgct tgaactagta
      181 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
      241 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
      301 agtcgatata acttcgtata gcatacatta tacgaagtta tgtcgaggag ggcctatttc
      361 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
      421 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
      481 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
      541 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgata
      601 acttcgtata gcatacatta tacgaagtta tggtaccgat gcagctagcc cgcggatctg
      661 ccggtctccc tatagtgagt cgtattaatt tcgataagcc aggttaacct gcattaatga
      721 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
      781 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
      841 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
      901 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
      961 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     1021 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     1081 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     1141 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     1201 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     1261 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     1321 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     1381 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     1441 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     1501 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     1561 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     1621 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     1681 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     1741 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     1801 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     1861 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     1921 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     1981 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     2041 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     2101 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     2161 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     2221 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     2281 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     2341 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     2401 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     2461 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     2521 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     2581 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     2641 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     2701 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     2761 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     2821 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     2881 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     2941 gtgcaccata tggacatatt gtcgttagaa cgcggctaca attaatacat aaccttatgt
     3001 atcatacaca tacg
//
