LOCUS       sequence-232665-        4573 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4573)
  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 4573)
  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-Z13
FEATURES             Location/Qualifiers
     source          1..4573
                     /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..788
                     /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"
     polyA_signal    795..916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(832..851)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     886..905
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(923..956)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        963..1279
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1130..1265
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1286..1295
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1292..1666
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    1676..1900
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1907..2155
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1907..1927
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2078..2097
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2157..2190)
                     /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(2227..2246)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2228..2246)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2333..2350)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2504..3092)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2584..2603)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3263..4054,4055..4123))
                     /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     3886..3905
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4124..4228)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4296..4314
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4352..4374)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4474..4493
                     /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 acaagtgaac tagtaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
      841 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
      901 tcatcaatgt atcttagtcg atataacttc gtatagcata cattatacga agttatgtcg
      961 acggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     1021 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     1081 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     1141 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     1201 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     1261 aggcctaggc ttttgcaaag aattcgccac catggccaag ttgaccagtg ccgttccggt
     1321 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc
     1381 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat
     1441 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg
     1501 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc
     1561 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga
     1621 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgactgc agcgactgtg
     1681 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1741 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     1801 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     1861 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg ctcgaggagg gcctatttcc
     1921 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     1981 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     2041 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     2101 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgataa
     2161 cttcgtatag catacattat acgaagttat ggtaccgatg cagctagccc gcggatctgc
     2221 cggtctccct atagtgagtc gtattaattt cgataagcca ggttaacctg cattaatgaa
     2281 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     2341 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     2401 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     2461 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2521 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2581 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2641 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2701 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2761 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2821 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     2881 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2941 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3001 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3061 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3121 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3181 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     3241 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     3301 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     3361 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     3421 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     3481 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     3541 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     3601 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     3661 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     3721 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     3781 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     3841 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     3901 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     3961 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4021 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4081 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4141 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4201 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     4261 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     4321 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     4381 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     4441 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
     4501 tgcaccatat ggacatattg tcgttagaac gcggctacaa ttaatacata accttatgta
     4561 tcatacacat acg
//
