LOCUS       Exported                4159 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with 3xFLAG.
ACCESSION   .
VERSION     .
KEYWORDS    pMaCTag-P34
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4159)
  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 4159)
  AUTHORS   .
  TITLE     Direct Submission
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"
FEATURES             Location/Qualifiers
     source          1..4159
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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..146
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    156..277
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(193..212)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     247..266
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    284..317
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        324..640
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      491..626
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     553..572
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    1262..1486
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1493..1733
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     1493..1513
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1664..1683
                     /label=LKO.1 5
                     /note="Human U6 promoter, forward primer"
     protein_bind    1743..1776
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(1813..1832)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1814..1832)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1919..1936)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2090..2678)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2170..2189)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2849..3709)
                     /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     3472..3491
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3710..3814)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3882..3900
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3938..3960)
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     primer_bind     4060..4079
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"