LOCUS       sequence-232662-        4519 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with mKOk.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4519)
  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 4519)
  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-Z10
FEATURES             Location/Qualifiers
     source          1..4519
                     /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..734
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="VSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVT
                     MAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSAS
                     VSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYL
                     KLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     polyA_signal    741..862
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(778..797)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     832..851
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(869..902)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        909..1225
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1076..1211
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1232..1241
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1238..1612
                     /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    1622..1846
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1853..2101
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1853..1873
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2024..2043
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2103..2136)
                     /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(2173..2192)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2174..2192)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2279..2296)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2450..3038)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2530..2549)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3209..4000,4001..4069))
                     /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     3832..3851
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4070..4174)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4242..4260
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4298..4320)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4420..4439
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagtgtga ttaaaccaga gatgaagatg aggtactaca
      121 tggacggctc cgtcaatggg catgagttca caattgaagg tgaaggcaca ggcagacctt
      181 acgagggaca tcaagagatg acactacgcg tcacaatggc cgaaggcggg ccaatgcctt
      241 tcgcgtttga cttagtgtca cacgtgttct gttacggcca cagagttttt actaaatatc
      301 cagaagagat accagactat ttcaaacaag catttcctga aggcctgtca tgggaaaggt
      361 cgttggagtt cgaagatggt gggtccgctt cagtcagtgc gcatataagc cttagaggaa
      421 acaccttcta ccacaaatcc aaatttactg gggttaactt tcctgccgat ggtcctatca
      481 tgcaaaacca aagtgttgat tgggagccat caaccgagaa aattactgcc agcgacggag
      541 ttctgaaggg tgatgttacg atgtacctaa aacttgaagg aggcggcaat cacaaatgcc
      601 aattcaagac tacttacaag gcggcaaaag aaattcttga aatgccagga gatcattaca
      661 tcggtcatcg cctcgtcagg aaaaccgaag gcaacattac tgagcaagta gaagatgcag
      721 tagctcattc ctgaactagt aacttgttta ttgcagctta taatggttac aaataaagca
      781 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
      841 ccaaactcat caatgtatct tagtcgatat aacttcgtat agcatacatt atacgaagtt
      901 atgtcgacgg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
      961 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     1021 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     1081 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     1141 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     1201 ttttggaggc ctaggctttt gcaaagaatt cgccaccatg gccaagttga ccagtgccgt
     1261 tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg
     1321 gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct
     1381 gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt
     1441 gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga
     1501 cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct
     1561 gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact gactgcagcg
     1621 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     1681 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     1741 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     1801 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggctcg aggagggcct
     1861 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattgg
     1921 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     1981 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
     2041 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
     2101 cgataacttc gtatagcata cattatacga agttatggta ccgatgcagc tagcccgcgg
     2161 atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt aacctgcatt
     2221 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2281 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2341 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2401 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2461 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2521 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2581 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2641 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2701 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2761 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     2821 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     2881 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     2941 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3001 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3061 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3121 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3181 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3241 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3301 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3361 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3421 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3481 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3541 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3601 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3661 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3721 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     3781 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     3841 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     3901 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     3961 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4021 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4081 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4141 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4201 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     4261 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     4321 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     4381 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     4441 tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa tacataacct
     4501 tatgtatcat acacatacg
//
