LOCUS       sequence-228128-        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"
     CDS             64..717
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="VSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVT
                     MAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSAS
                     VSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYL
                     KLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     polyA_signal    724..845
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(761..780)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     815..834
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(852..885)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        892..1208
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1059..1194
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1215..1224
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1221..1595
                     /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    1605..1829
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1836..2084
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1836..1856
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2007..2026
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2086..2119)
                     /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(2156..2175)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2157..2175)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2262..2279)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2433..3021)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2513..2532)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3192..3983,3984..4052))
                     /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     3815..3834
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4053..4157)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4225..4243
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4281..4303)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4403..4422
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4503..4519,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4503..4519,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagtg tgattaaacc agagatgaag atgaggtact acatggacgg ctccgtcaat
      121 gggcatgagt tcacaattga aggtgaaggc acaggcagac cttacgaggg acatcaagag
      181 atgacactac gcgtcacaat ggccgaaggc gggccaatgc ctttcgcgtt tgacttagtg
      241 tcacacgtgt tctgttacgg ccacagagtt tttactaaat atccagaaga gataccagac
      301 tatttcaaac aagcatttcc tgaaggcctg tcatgggaaa ggtcgttgga gttcgaagat
      361 ggtgggtccg cttcagtcag tgcgcatata agccttagag gaaacacctt ctaccacaaa
      421 tccaaattta ctggggttaa ctttcctgcc gatggtccta tcatgcaaaa ccaaagtgtt
      481 gattgggagc catcaaccga gaaaattact gccagcgacg gagttctgaa gggtgatgtt
      541 acgatgtacc taaaacttga aggaggcggc aatcacaaat gccaattcaa gactacttac
      601 aaggcggcaa aagaaattct tgaaatgcca ggagatcatt acatcggtca tcgcctcgtc
      661 aggaaaaccg aaggcaacat tactgagcaa gtagaagatg cagtagctca ttcctgaact
      721 agtaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
      781 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
      841 tcttagtcga tataacttcg tatagcatac attatacgaa gttatgtcga cggtgtggaa
      901 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
      961 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     1021 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     1081 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     1141 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     1201 tttgcaaaga attcgccacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc
     1261 gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg
     1321 tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc
     1381 aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc
     1441 tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca
     1501 tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca
     1561 actgcgtgca cttcgtggcc gaggagcagg actgactgca gcgactgtgc cttctagttg
     1621 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     1681 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     1741 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     1801 gcatgctggg gatgcggtgg gctctatggc tcgaggaggg cctatttccc atgattcctt
     1861 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     1921 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     1981 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     2041 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgataac ttcgtatagc
     2101 atacattata cgaagttatg gtaccgatgc agctagcccg cggatctgcc ggtctcccta
     2161 tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat cggccaacgc
     2221 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     2281 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     2341 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     2401 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2461 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2521 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2581 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt
     2641 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2701 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     2761 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     2821 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     2881 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     2941 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3001 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3061 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3121 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     3181 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     3241 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     3301 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     3361 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     3421 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     3481 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     3541 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     3601 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     3661 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     3721 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     3781 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     3841 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     3901 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     3961 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4021 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4081 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     4141 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     4201 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt
     4261 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     4321 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     4381 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
     4441 gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat catacacata
     4501 cgatttaggt gacactata
//
