LOCUS       sequence-232596-        3587 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with mKeima8.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3587)
  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 3587)
  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-18
FEATURES             Location/Qualifiers
     source          1..3587
                     /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             join(81..83,84..743)
                     /codon_start=1
                     /product="human codon-optimized monomeric red fluorescent 
                     protein with a large Stokes shift (Guan et al. 2015)"
                     /label=hmKeima8.5
                     /translation="VSVIAEHMTYKVYMSGTVNGHYFEVEGDGEGKPYEGEQTVKLTVT
                     KGGPLPFAWHILSPQLQYGSIPFTKYPADIPDYFKQSFPEGYTWVRSMNFEDGAVCTVS
                     NDSSIRGNCFIYNVKISGENFPPNGPVMQKKTQGWEPSTERMFARDGMLIGNDHMALKL
                     EGGGHYLCEFKSAYKARKPVRMPGLYEIDRKLDVTSHNRDYTSVEQREIAIARHPLLG"
     polyA_signal    753..874
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(790..809)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     844..863
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(881..914)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        921..1169
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     921..941
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1092..1111
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1171..1204)
                     /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(1241..1260)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1242..1260)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1347..1364)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1518..2106)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1598..1617)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2277..3068,3069..3137))
                     /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     2900..2919
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3138..3242)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3310..3328
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3366..3388)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3488..3507
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagcgtga tcgccgagca catgacctac aaggtgtaca
      121 tgagcggcac cgtgaacggc cactacttcg aggtggaggg cgacggcgag ggcaagccct
      181 acgagggcga gcagaccgtg aagctgaccg tgaccaaggg tggccccctg cccttcgcct
      241 ggcacatcct gagcccccag ctccagtacg gcagcatccc cttcaccaag taccccgccg
      301 acatccccga ctacttcaag cagagcttcc ccgagggcta cacctgggtg cgcagcatga
      361 acttcgagga cggcgccgtg tgcaccgtga gcaacgacag cagcatccgc ggcaactgct
      421 tcatctacaa cgtgaagatc agcggcgaga acttcccccc caacggcccc gtgatgcaga
      481 agaagaccca gggctgggag cccagcaccg agcgcatgtt cgcccgcgac ggaatgctga
      541 tcggcaacga ccacatggcc ctgaagctgg agggcggcgg ccactacctg tgcgagttca
      601 agagcgccta caaggcccgc aagcccgtga ggatgcccgg cctgtacgag atcgaccgca
      661 agctggacgt gaccagccac aaccgcgact acaccagcgt ggagcagcgc gagatcgcca
      721 tcgcccgcca ccccctgctg ggctgaacta gtaacttgtt tattgcagct tataatggtt
      781 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
      841 gttgtggttt gtccaaactc atcaatgtat cttagtcgat ataacttcgt atagcataca
      901 ttatacgaag ttatgtcgag gagggcctat ttcccatgat tccttcatat ttgcatatac
      961 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     1021 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     1081 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     1141 tatatatctt gtggaaagga cgaaacaccg ataacttcgt atagcataca ttatacgaag
     1201 ttatggtacc gatgcagcta gcccgcggat ctgccggtct ccctatagtg agtcgtatta
     1261 atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     1321 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     1381 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     1441 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1501 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1561 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1621 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     1681 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     1741 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     1801 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     1861 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     1921 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     1981 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2041 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     2101 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2161 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2221 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2281 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2341 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     2401 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     2461 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     2521 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     2581 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     2641 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     2701 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     2761 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     2821 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     2881 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     2941 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3001 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     3061 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     3121 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     3181 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3241 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     3301 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     3361 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     3421 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     3481 aactatgcgg catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta
     3541 gaacgcggct acaattaata cataacctta tgtatcatac acatacg
//
