LOCUS       sequence-228016-        3629 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3629)
  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 3629)
  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-13
FEATURES             Location/Qualifiers
     source          1..3629
                     /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..1194
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     946..966
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1117..1136
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1196..1229)
                     /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(1266..1285)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1267..1285)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1372..1389)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1543..2131)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1623..1642)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2302..3093,3094..3162))
                     /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     2925..2944
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3163..3267)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3335..3353
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3391..3413)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3513..3532
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3613..3629,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3613..3629,1)
                     /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 tcgaggaggg cctatttccc
      961 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     1021 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     1081 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     1141 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgataac
     1201 ttcgtatagc atacattata cgaagttatg gtaccgatgc agctagcccg cggatctgcc
     1261 ggtctcccta tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat
     1321 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     1381 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     1441 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1501 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1561 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1621 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1681 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg
     1741 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1801 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     1861 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     1921 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     1981 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2041 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2101 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2161 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2221 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2281 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2341 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2401 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     2461 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     2521 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     2581 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     2641 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     2701 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     2761 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     2821 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     2881 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     2941 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     3001 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     3061 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     3121 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     3181 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3241 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     3301 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     3361 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     3421 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     3481 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     3541 gcaccatatg gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat
     3601 catacacata cgatttaggt gacactata
//
