LOCUS       sequence-228014-        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"
     CDS             64..756
                     /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(194..212)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     462..481
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    766..887
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(803..822)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     857..876
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(894..927)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        934..1182
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     934..954
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1105..1124
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1184..1217)
                     /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(1254..1273)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1255..1273)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1360..1377)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1531..2119)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1611..1630)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2290..3081,3082..3150))
                     /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     2913..2932
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3151..3255)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3323..3341
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3379..3401)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3501..3520
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3601..3617,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3601..3617,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagca agggcgaggc agtgatcaag gagttcatgc ggttcaaggt gcacatggag
      121 ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
      181 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt ctcctgggac
      241 atcctgtccc ctcagttcat gtacggctcc agggccttca tcaagcaccc cgccgacatc
      301 cccgactact ataagcagtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
      361 gaggacggcg gcgccgtgac cgtgacccag gacacctccc tggaggacgg caccctgatc
      421 tacaaggtga agctccgcgg caccaacttc cctcctgacg gccccgtaat gcagaagaag
      481 acaatgggct gggaagcgtc caccgagcgg ttgtaccccg aggacggcgt gctgaagggc
      541 gacattaaga tggccctgcg cctgaaggac ggcggccgct acctggcgga cttcaagacc
      601 acctacaagg ccaagaagcc cgtgcagatg cccggcgcct acaacgtcga ccgcaagttg
      661 gacatcacct cccacaacga ggactacacc gtggtggaac agtacgaacg ctccgagggc
      721 cgccactcca ccggcggcat ggacgagctg tacaagtgaa ctagtaactt gtttattgca
      781 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
      841 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttagtc gatataactt
      901 cgtatagcat acattatacg aagttatgtc gaggagggcc tatttcccat gattccttca
      961 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
     1021 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     1081 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     1141 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgataactt cgtatagcat
     1201 acattatacg aagttatggt accgatgcag ctagcccgcg gatctgccgg tctccctata
     1261 gtgagtcgta ttaatttcga taagccaggt taacctgcat taatgaatcg gccaacgcgc
     1321 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     1381 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     1441 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     1501 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     1561 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     1621 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     1681 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag
     1741 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     1801 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     1861 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     1921 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     1981 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     2041 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     2101 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     2161 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     2221 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     2281 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     2341 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     2401 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     2461 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     2521 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     2581 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     2641 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     2701 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     2761 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     2821 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     2881 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     2941 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     3001 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     3061 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     3121 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     3181 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     3241 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     3301 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
     3361 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     3421 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     3481 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgga
     3541 catattgtcg ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg
     3601 atttaggtga cactata
//
