LOCUS       sequence-232630-        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"
     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..788
                     /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(223..241)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     491..510
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    795..916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(832..851)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     886..905
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(923..956)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        963..1279
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1130..1265
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1901..2125
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2132..2380
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2132..2152
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2303..2322
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2382..2415)
                     /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(2452..2471)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2453..2471)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2558..2575)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2729..3317)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2809..2828)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3488..4279,4280..4348))
                     /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     4111..4130
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4349..4453)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4521..4539
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4577..4599)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4699..4718
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggagga taacatggcc atcatcaagg
      121 agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg
      181 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
      241 agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca
      301 aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg
      361 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
      421 actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc
      481 cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga
      541 tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg
      601 gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc
      661 ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca
      721 tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt
      781 acaagtgaac tagtaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
      841 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
      901 tcatcaatgt atcttagtcg atataacttc gtatagcata cattatacga agttatgtcg
      961 acggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     1021 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     1081 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     1141 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     1201 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     1261 aggcctaggc ttttgcaaag aattcgccac catgactgag tacaagccta ctgtgcgttt
     1321 agccaccagg gatgatgtgc cacgagccgt gaggacacta gcagccgcct ttgctgacta
     1381 cccagctaca aggcacaccg tggaccctga cagacacatt gaaagagtca cagaacttca
     1441 ggagctgttt ttaactaggg tgggcctgga tattggcaaa gtctgggtgg ccgatgatgg
     1501 agcagcagtt gcagtgtgga ccaccccaga gtctgttgaa gctggagctg tctttgcaga
     1561 aattggacca agaatggccg agttatctgg cagcaggctg gctgctcaac agcagatgga
     1621 aggattatta gctccccaca gacccaagga gcctgcctgg ttcctggcaa cagttggagt
     1681 ttctccagac catcagggca agggcttagg atctgctgtg gtgctgcctg gtgtggaagc
     1741 agctgagaga gctggggtgc cagcatttct ggagacatca gctcctagaa acctgccctt
     1801 ctatgaacgg ctgggcttca cagtgactgc tgatgtggag gtgcctgagg gccccagaac
     1861 ctggtgcatg acaagaaaac caggagcctg actgcagcga ctgtgccttc tagttgccag
     1921 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     1981 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     2041 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     2101 gctggggatg cggtgggctc tatggctcga ggagggccta tttcccatga ttccttcata
     2161 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     2221 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     2281 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     2341 tttcttggct ttatatatct tgtggaaagg acgaaacacc gataacttcg tatagcatac
     2401 attatacgaa gttatggtac cgatgcagct agcccgcgga tctgccggtc tccctatagt
     2461 gagtcgtatt aatttcgata agccaggtta acctgcatta atgaatcggc caacgcgcgg
     2521 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     2581 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     2641 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     2701 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2761 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2821 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2881 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     2941 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3001 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3061 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3121 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     3181 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3241 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3301 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3361 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     3421 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3481 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3541 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     3601 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3661 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3721 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3781 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3841 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3901 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3961 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4021 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4081 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4141 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4201 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     4261 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     4321 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     4381 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     4441 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     4501 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     4561 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     4621 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     4681 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatggaca
     4741 tattgtcgtt agaacgcggc tacaattaat acataacctt atgtatcata cacatacg
//
