LOCUS       sequence-228131-        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"
     CDS             64..771
                     /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(206..224)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     474..493
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    778..899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(815..834)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     869..888
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(906..939)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        946..1262
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1113..1248
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1269..1278
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1275..1649
                     /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    1659..1883
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1890..2138
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1890..1910
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2061..2080
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2140..2173)
                     /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(2210..2229)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2211..2229)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2316..2333)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2487..3075)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2567..2586)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3246..4037,4038..4106))
                     /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     3869..3888
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4107..4211)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4279..4297
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4335..4357)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4457..4476
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4557..4573,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4557..4573,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
      121 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
      181 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
      241 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
      301 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
      361 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
      421 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
      481 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
      541 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
      601 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
      661 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
      721 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagtg aactagtaac
      781 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
      841 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttag
      901 tcgatataac ttcgtatagc atacattata cgaagttatg tcgacggtgt ggaaagtccc
      961 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     1021 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     1081 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     1141 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     1201 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     1261 aagaattcgc caccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg
     1321 tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg
     1381 acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc
     1441 aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg
     1501 ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg
     1561 agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg
     1621 tgcacttcgt ggccgaggag caggactgac tgcagcgact gtgccttcta gttgccagcc
     1681 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     1741 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     1801 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     1861 tggggatgcg gtgggctcta tggctcgagg agggcctatt tcccatgatt ccttcatatt
     1921 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     1981 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     2041 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     2101 tcttggcttt atatatcttg tggaaaggac gaaacaccga taacttcgta tagcatacat
     2161 tatacgaagt tatggtaccg atgcagctag cccgcggatc tgccggtctc cctatagtga
     2221 gtcgtattaa tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg
     2281 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     2341 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2401 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2461 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2521 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     2581 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2641 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat
     2701 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2761 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2821 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2881 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     2941 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     3001 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3061 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3121 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3181 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     3241 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3301 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     3361 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     3421 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3481 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     3541 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     3601 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3661 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     3721 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     3781 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3841 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     3901 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3961 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     4021 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     4081 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     4141 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4201 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     4261 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     4321 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     4381 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     4441 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatggacata
     4501 ttgtcgttag aacgcggcta caattaatac ataaccttat gtatcataca catacgattt
     4561 aggtgacact ata
//
