LOCUS       sequence-243813-        4367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with mCherry-mNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4367)
  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 4367)
  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-19
FEATURES             Location/Qualifiers
     source          1..4367
                     /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..785
                     /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"
     CDS             816..1526
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      1071..1080
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1533..1654
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1570..1589)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1624..1643
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1661..1694)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1701..1949
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1701..1721
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1872..1891
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1951..1984)
                     /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(2021..2040)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2022..2040)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2127..2144)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2298..2886)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2378..2397)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3057..3848,3849..3917))
                     /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     3680..3699
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3918..4022)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4090..4108
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4146..4168)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4268..4287
                     /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 acaagggtgc aggagctgga accggtgcag gagctatggt gagcaagggc gaggaggata
      841 acatggcctc tctcccagcg acacatgagt tacacatctt tggctccatc aacggtgtgg
      901 actttgacat ggtgggtcag ggcaccggca atccaaatga tggttatgag gagttaaacc
      961 tgaagtccac caagggtgac ctccagttct ccccctggat tctggtccct catatcgggt
     1021 atggcttcca tcagtacctg ccctaccctg acgggatgtc gcctttccag gccgccatgg
     1081 tagatggctc cggataccaa gtccatcgca caatgcagtt tgaagatggt gcctccctta
     1141 ctgttaacta ccgctacacc tacgagggaa gccacatcaa aggagaggcc caggtgaagg
     1201 ggactggttt ccctgctgac ggtcctgtga tgaccaactc gctgaccgct gcggactggt
     1261 gcaggtcgaa gaagacttac cccaacgaca aaaccatcat cagtaccttt aagtggagtt
     1321 acaccactgg aaatggcaag cgctaccgga gcactgcgcg gaccacctac acctttgcca
     1381 agccaatggc ggctaactat ctgaagaacc agccgatgta cgtgttccgt aagacggagc
     1441 tcaagcactc caagaccgag ctcaacttca aggagtggca aaaggccttt accgatgtga
     1501 tgggcatgga cgagctgtac aagtgaacta gtaacttgtt tattgcagct tataatggtt
     1561 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     1621 gttgtggttt gtccaaactc atcaatgtat cttagtcgat ataacttcgt atagcataca
     1681 ttatacgaag ttatgtcgag gagggcctat ttcccatgat tccttcatat ttgcatatac
     1741 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     1801 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     1861 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     1921 tatatatctt gtggaaagga cgaaacaccg ataacttcgt atagcataca ttatacgaag
     1981 ttatggtacc gatgcagcta gcccgcggat ctgccggtct ccctatagtg agtcgtatta
     2041 atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2101 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2161 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2221 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2281 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2341 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2401 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2461 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     2521 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2581 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2641 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2701 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     2761 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2821 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     2881 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2941 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3001 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3061 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3121 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3181 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     3241 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3301 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3361 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3421 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3481 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3541 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3601 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3661 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3721 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3781 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     3841 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     3901 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     3961 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4021 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     4081 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     4141 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     4201 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     4261 aactatgcgg catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta
     4321 gaacgcggct acaattaata cataacctta tgtatcatac acatacg
//
