LOCUS       sequence-228021-        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"
     CDS             join(64..66,67..726)
                     /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    736..857
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(773..792)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     827..846
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(864..897)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        904..1152
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     904..924
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1075..1094
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1154..1187)
                     /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(1224..1243)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1225..1243)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1330..1347)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1501..2089)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1581..1600)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2260..3051,3052..3120))
                     /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     2883..2902
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3121..3225)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3293..3311
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3349..3371)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3471..3490
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3571..3587,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3571..3587,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagcg tgatcgccga gcacatgacc tacaaggtgt acatgagcgg caccgtgaac
      121 ggccactact tcgaggtgga gggcgacggc gagggcaagc cctacgaggg cgagcagacc
      181 gtgaagctga ccgtgaccaa gggtggcccc ctgcccttcg cctggcacat cctgagcccc
      241 cagctccagt acggcagcat ccccttcacc aagtaccccg ccgacatccc cgactacttc
      301 aagcagagct tccccgaggg ctacacctgg gtgcgcagca tgaacttcga ggacggcgcc
      361 gtgtgcaccg tgagcaacga cagcagcatc cgcggcaact gcttcatcta caacgtgaag
      421 atcagcggcg agaacttccc ccccaacggc cccgtgatgc agaagaagac ccagggctgg
      481 gagcccagca ccgagcgcat gttcgcccgc gacggaatgc tgatcggcaa cgaccacatg
      541 gccctgaagc tggagggcgg cggccactac ctgtgcgagt tcaagagcgc ctacaaggcc
      601 cgcaagcccg tgaggatgcc cggcctgtac gagatcgacc gcaagctgga cgtgaccagc
      661 cacaaccgcg actacaccag cgtggagcag cgcgagatcg ccatcgcccg ccaccccctg
      721 ctgggctgaa ctagtaactt gtttattgca gcttataatg gttacaaata aagcaatagc
      781 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
      841 ctcatcaatg tatcttagtc gatataactt cgtatagcat acattatacg aagttatgtc
      901 gaggagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
      961 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     1021 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     1081 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     1141 ggacgaaaca ccgataactt cgtatagcat acattatacg aagttatggt accgatgcag
     1201 ctagcccgcg gatctgccgg tctccctata gtgagtcgta ttaatttcga taagccaggt
     1261 taacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     1321 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     1381 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     1441 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     1501 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     1561 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     1621 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     1681 cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     1741 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     1801 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     1861 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     1921 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     1981 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2041 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2101 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     2161 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     2221 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     2281 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     2341 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     2401 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     2461 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     2521 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     2581 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     2641 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     2701 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     2761 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     2821 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     2881 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     2941 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     3001 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     3061 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     3121 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     3181 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     3241 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     3301 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     3361 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     3421 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     3481 gcagattgta ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta
     3541 atacataacc ttatgtatca tacacatacg atttaggtga cactata
//
