LOCUS       sequence-232603-        2951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with 1xHA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2951)
  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 2951)
  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-27
FEATURES             Location/Qualifiers
     source          1..2951
                     /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..533
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     285..305
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     456..475
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(535..568)
                     /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(605..624)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(606..624)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(711..728)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(882..1470)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(962..981)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1641..2432,2433..2501))
                     /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     2264..2283
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2502..2606)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2674..2692
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2730..2752)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2852..2871
                     /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 cgaggagggc ctatttccca
      301 tgattccttc atatttgcat atacgataca aggctgttag agagataatt ggaattaatt
      361 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
      421 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
      481 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgataact
      541 tcgtatagca tacattatac gaagttatgg taccgatgca gctagcccgc ggatctgccg
      601 gtctccctat agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc
      661 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
      721 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
      781 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
      841 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
      901 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
      961 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     1021 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     1081 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     1141 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     1201 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     1261 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     1321 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     1381 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     1441 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     1501 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     1561 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     1621 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     1681 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     1741 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     1801 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     1861 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     1921 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     1981 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     2041 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     2101 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     2161 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     2221 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     2281 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     2341 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     2401 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     2461 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     2521 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     2581 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     2641 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     2701 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     2761 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     2821 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
     2881 caccatatgg acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc
     2941 atacacatac g
//
