LOCUS       sequence-228055-        4798 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4798)
  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 4798)
  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-P13
FEATURES             Location/Qualifiers
     source          1..4798
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..771
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(206..224)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     474..493
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    778..899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(815..834)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     869..888
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(906..939)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        946..1262
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1113..1248
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1884..2108
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2115..2363
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2115..2135
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2286..2305
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2365..2398)
                     /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(2435..2454)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2436..2454)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2541..2558)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2712..3300)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2792..2811)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3471..4262,4263..4331))
                     /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     4094..4113
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4332..4436)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4504..4522
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4560..4582)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4682..4701
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4782..4798,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4782..4798,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
      121 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
      181 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
      241 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
      301 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
      361 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
      421 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
      481 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
      541 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
      601 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
      661 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
      721 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagtg aactagtaac
      781 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
      841 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttag
      901 tcgatataac ttcgtatagc atacattata cgaagttatg tcgacggtgt ggaaagtccc
      961 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     1021 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     1081 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     1141 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     1201 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     1261 aagaattcgc caccatgact gagtacaagc ctactgtgcg tttagccacc agggatgatg
     1321 tgccacgagc cgtgaggaca ctagcagccg cctttgctga ctacccagct acaaggcaca
     1381 ccgtggaccc tgacagacac attgaaagag tcacagaact tcaggagctg tttttaacta
     1441 gggtgggcct ggatattggc aaagtctggg tggccgatga tggagcagca gttgcagtgt
     1501 ggaccacccc agagtctgtt gaagctggag ctgtctttgc agaaattgga ccaagaatgg
     1561 ccgagttatc tggcagcagg ctggctgctc aacagcagat ggaaggatta ttagctcccc
     1621 acagacccaa ggagcctgcc tggttcctgg caacagttgg agtttctcca gaccatcagg
     1681 gcaagggctt aggatctgct gtggtgctgc ctggtgtgga agcagctgag agagctgggg
     1741 tgccagcatt tctggagaca tcagctccta gaaacctgcc cttctatgaa cggctgggct
     1801 tcacagtgac tgctgatgtg gaggtgcctg agggccccag aacctggtgc atgacaagaa
     1861 aaccaggagc ctgactgcag cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     1921 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     1981 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2041 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     2101 ctctatggct cgaggagggc ctatttccca tgattccttc atatttgcat atacgataca
     2161 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     2221 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     2281 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     2341 tcttgtggaa aggacgaaac accgataact tcgtatagca tacattatac gaagttatgg
     2401 taccgatgca gctagcccgc ggatctgccg gtctccctat agtgagtcgt attaatttcg
     2461 ataagccagg ttaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2521 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     2581 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     2641 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     2701 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2761 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     2821 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     2881 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag
     2941 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3001 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3061 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3121 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     3181 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3241 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     3301 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     3361 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     3421 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     3481 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     3541 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     3601 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     3661 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3721 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3781 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3841 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3901 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3961 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4021 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4081 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4141 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4201 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4261 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     4321 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     4381 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     4441 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     4501 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     4561 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     4621 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     4681 gcggcatcag agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc
     4741 ggctacaatt aatacataac cttatgtatc atacacatac gatttaggtg acactata
//
