LOCUS       sequence-232508-        3617 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with mScarlet-i.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3617)
  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 3617)
  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-11
FEATURES             Location/Qualifiers
     source          1..3617
                     /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..773
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(211..229)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     479..498
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    783..904
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(820..839)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     874..893
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(911..944)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        951..1199
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     951..971
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1122..1141
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1201..1234)
                     /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(1271..1290)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1272..1290)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1377..1394)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1548..2136)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1628..1647)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2307..3098,3099..3167))
                     /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     2930..2949
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3168..3272)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3340..3358
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3396..3418)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3518..3537
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggcagt gatcaaggag ttcatgcggt
      121 tcaaggtgca catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg
      181 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
      241 tgcccttctc ctgggacatc ctgtcccctc agttcatgta cggctccagg gccttcatca
      301 agcaccccgc cgacatcccc gactactata agcagtcctt ccccgagggc ttcaagtggg
      361 agcgcgtgat gaacttcgag gacggcggcg ccgtgaccgt gacccaggac acctccctgg
      421 aggacggcac cctgatctac aaggtgaagc tccgcggcac caacttccct cctgacggcc
      481 ccgtaatgca gaagaagaca atgggctggg aagcgtccac cgagcggttg taccccgagg
      541 acggcgtgct gaagggcgac attaagatgg ccctgcgcct gaaggacggc ggccgctacc
      601 tggcggactt caagaccacc tacaaggcca agaagcccgt gcagatgccc ggcgcctaca
      661 acgtcgaccg caagttggac atcacctccc acaacgagga ctacaccgtg gtggaacagt
      721 acgaacgctc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtgaacta
      781 gtaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
      841 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
      901 cttagtcgat ataacttcgt atagcataca ttatacgaag ttatgtcgag gagggcctat
      961 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     1021 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     1081 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     1141 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
     1201 ataacttcgt atagcataca ttatacgaag ttatggtacc gatgcagcta gcccgcggat
     1261 ctgccggtct ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa
     1321 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     1381 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     1441 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     1501 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     1561 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     1621 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     1681 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     1741 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     1801 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1861 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     1921 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     1981 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2041 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2101 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2161 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     2221 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2281 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     2341 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     2401 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     2461 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     2521 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     2581 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     2641 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     2701 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     2761 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     2821 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     2881 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     2941 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3001 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3061 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3121 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3181 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3241 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     3301 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     3361 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     3421 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     3481 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     3541 agagtgcacc atatggacat attgtcgtta gaacgcggct acaattaata cataacctta
     3601 tgtatcatac acatacg
//
