LOCUS       sequence-227997-        2948 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2948)
  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 2948)
  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-33
FEATURES             Location/Qualifiers
     source          1..2948
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..87
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    97..218
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(134..153)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     188..207
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(225..258)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        265..513
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     265..285
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     436..455
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(515..548)
                     /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(585..604)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(586..604)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(691..708)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(862..1450)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(942..961)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1621..2412,2413..2481))
                     /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     2244..2263
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2482..2586)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2654..2672
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2710..2732)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2832..2851
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(2932..2948,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(2932..2948,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgattaca aggatgacga tgacaagtga actagtaact tgtttattgc agcttataat
      121 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
      181 tctagttgtg gtttgtccaa actcatcaat gtatcttagt cgatataact tcgtatagca
      241 tacattatac gaagttatgt cgaggagggc ctatttccca tgattccttc atatttgcat
      301 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      361 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      421 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      481 gctttatata tcttgtggaa aggacgaaac accgataact tcgtatagca tacattatac
      541 gaagttatgg taccgatgca gctagcccgc ggatctgccg gtctccctat agtgagtcgt
      601 attaatttcg ataagccagg ttaacctgca ttaatgaatc ggccaacgcg cggggagagg
      661 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
      721 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
      781 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
      841 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
      901 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
      961 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     1021 cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag
     1081 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     1141 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     1201 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     1261 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     1321 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     1381 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     1441 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     1501 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     1561 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     1621 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     1681 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     1741 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     1801 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     1861 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     1921 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     1981 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     2041 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     2101 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     2161 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     2221 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     2281 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     2341 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     2401 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     2461 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     2521 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     2581 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     2641 attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga
     2701 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     2761 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
     2821 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgg acatattgtc
     2881 gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac gatttaggtg
     2941 acactata
//
