LOCUS       sequence-232664-        4561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with mScarlet.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4561)
  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 4561)
  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-Z12
FEATURES             Location/Qualifiers
     source          1..4561
                     /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
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     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..1267
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1118..1253
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1274..1283
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1280..1654
                     /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    1664..1888
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1895..2143
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1895..1915
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2066..2085
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2145..2178)
                     /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(2215..2234)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2216..2234)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2321..2338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2492..3080)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2572..2591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3251..4042,4043..4111))
                     /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     3874..3893
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4112..4216)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4284..4302
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4340..4362)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4462..4481
                     /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 gccttcacca
      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 ttatgtcgac ggtgtggaaa
      961 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     1021 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     1081 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     1141 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     1201 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     1261 ttgcaaagaa ttcgccacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
     1321 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
     1381 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
     1441 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
     1501 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
     1561 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
     1621 ctgcgtgcac ttcgtggccg aggagcagga ctgactgcag cgactgtgcc ttctagttgc
     1681 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     1741 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     1801 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     1861 catgctgggg atgcggtggg ctctatggct cgaggagggc ctatttccca tgattccttc
     1921 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
     1981 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     2041 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     2101 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgataact tcgtatagca
     2161 tacattatac gaagttatgg taccgatgca gctagcccgc ggatctgccg gtctccctat
     2221 agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc ggccaacgcg
     2281 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2341 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2401 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2461 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2521 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2581 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2641 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2701 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2761 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2821 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2881 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2941 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3001 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3061 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3121 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3181 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3241 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3301 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3361 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3421 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3481 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3541 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3601 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3661 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3721 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3781 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3841 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3901 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3961 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4021 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4081 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4141 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4201 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     4261 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     4321 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     4381 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     4441 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgg
     4501 acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac
     4561 g
//
