LOCUS       sequence-232619-        4744 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with mKOk.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4744)
  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 4744)
  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-P10
FEATURES             Location/Qualifiers
     source          1..4744
                     /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"
     polyA_signal    1847..2071
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2078..2326
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2078..2098
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2249..2268
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2328..2361)
                     /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(2398..2417)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2399..2417)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2504..2521)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2675..3263)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2755..2774)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3434..4225,4226..4294))
                     /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     4057..4076
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4295..4399)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4467..4485
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4523..4545)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4645..4664
                     /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 actgagtaca agcctactgt
     1261 gcgtttagcc accagggatg atgtgccacg agccgtgagg acactagcag ccgcctttgc
     1321 tgactaccca gctacaaggc acaccgtgga ccctgacaga cacattgaaa gagtcacaga
     1381 acttcaggag ctgtttttaa ctagggtggg cctggatatt ggcaaagtct gggtggccga
     1441 tgatggagca gcagttgcag tgtggaccac cccagagtct gttgaagctg gagctgtctt
     1501 tgcagaaatt ggaccaagaa tggccgagtt atctggcagc aggctggctg ctcaacagca
     1561 gatggaagga ttattagctc cccacagacc caaggagcct gcctggttcc tggcaacagt
     1621 tggagtttct ccagaccatc agggcaaggg cttaggatct gctgtggtgc tgcctggtgt
     1681 ggaagcagct gagagagctg gggtgccagc atttctggag acatcagctc ctagaaacct
     1741 gcccttctat gaacggctgg gcttcacagt gactgctgat gtggaggtgc ctgagggccc
     1801 cagaacctgg tgcatgacaa gaaaaccagg agcctgactg cagcgactgt gccttctagt
     1861 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     1921 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     1981 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     2041 aggcatgctg gggatgcggt gggctctatg gctcgaggag ggcctatttc ccatgattcc
     2101 ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt
     2161 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     2221 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     2281 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgata acttcgtata
     2341 gcatacatta tacgaagtta tggtaccgat gcagctagcc cgcggatctg ccggtctccc
     2401 tatagtgagt cgtattaatt tcgataagcc aggttaacct gcattaatga atcggccaac
     2461 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2521 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2581 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2641 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2701 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2761 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2821 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2881 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2941 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3001 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3061 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     3121 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3181 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3241 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3301 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3361 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3421 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3481 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3541 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     3601 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3661 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3721 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     3781 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     3841 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     3901 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     3961 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     4021 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     4081 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     4141 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     4201 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     4261 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     4321 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4381 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     4441 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     4501 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     4561 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     4621 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     4681 tggacatatt gtcgttagaa cgcggctaca attaatacat aaccttatgt atcatacaca
     4741 tacg
//
