LOCUS       sequence-228045-        4774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with mClover3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4774)
  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 4774)
  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-P08
FEATURES             Location/Qualifiers
     source          1..4774
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(106..127)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     714..735
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    754..875
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(791..810)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     845..864
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(882..915)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        922..1238
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1089..1224
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1860..2084
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2091..2339
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2091..2111
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2262..2281
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2341..2374)
                     /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(2411..2430)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2412..2430)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2517..2534)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2688..3276)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2768..2787)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3447..4238,4239..4307))
                     /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     4070..4089
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4308..4412)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4480..4498
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4536..4558)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4658..4677
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4758..4774,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4758..4774,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      121 ggcgacgtaa acggccacaa gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac
      181 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      241 ctcgtgacca ccttcggcta cggcgtggcc tgcttcagcc gctaccccga ccacatgaag
      301 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctct
      361 ttcaaggacg acggtaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      421 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      481 aagctggagt acaacttcaa cagccactac gtctatatca cggccgacaa gcagaagaac
      541 tgcatcaagg ctaacttcaa gatccgccac aacgttgagg acggcagcgt gcagctcgcc
      601 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      661 tacctgagcc atcagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      721 ctgctggagt tcgtgaccgc cgcctgaact agtaacttgt ttattgcagc ttataatggt
      781 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
      841 agttgtggtt tgtccaaact catcaatgta tcttagtcga tataacttcg tatagcatac
      901 attatacgaa gttatgtcga cggtgtggaa agtccccagg ctccccagca ggcagaagta
      961 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     1021 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     1081 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     1141 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     1201 agtgaggagg cttttttgga ggcctaggct tttgcaaaga attcgccacc atgactgagt
     1261 acaagcctac tgtgcgttta gccaccaggg atgatgtgcc acgagccgtg aggacactag
     1321 cagccgcctt tgctgactac ccagctacaa ggcacaccgt ggaccctgac agacacattg
     1381 aaagagtcac agaacttcag gagctgtttt taactagggt gggcctggat attggcaaag
     1441 tctgggtggc cgatgatgga gcagcagttg cagtgtggac caccccagag tctgttgaag
     1501 ctggagctgt ctttgcagaa attggaccaa gaatggccga gttatctggc agcaggctgg
     1561 ctgctcaaca gcagatggaa ggattattag ctccccacag acccaaggag cctgcctggt
     1621 tcctggcaac agttggagtt tctccagacc atcagggcaa gggcttagga tctgctgtgg
     1681 tgctgcctgg tgtggaagca gctgagagag ctggggtgcc agcatttctg gagacatcag
     1741 ctcctagaaa cctgcccttc tatgaacggc tgggcttcac agtgactgct gatgtggagg
     1801 tgcctgaggg ccccagaacc tggtgcatga caagaaaacc aggagcctga ctgcagcgac
     1861 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1921 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1981 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2041 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggctcgag gagggcctat
     2101 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     2161 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     2221 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     2281 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
     2341 ataacttcgt atagcataca ttatacgaag ttatggtacc gatgcagcta gcccgcggat
     2401 ctgccggtct ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa
     2461 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     2521 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     2581 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2641 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2701 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2761 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2821 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2881 caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2941 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3001 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3061 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3121 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3181 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3241 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3301 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     3361 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3421 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3481 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3541 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     3601 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3661 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3721 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3781 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3841 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3901 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3961 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4021 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4081 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4141 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4201 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4261 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4321 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4381 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     4441 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     4501 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     4561 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     4621 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     4681 agagtgcacc atatggacat attgtcgtta gaacgcggct acaattaata cataacctta
     4741 tgtatcatac acatacgatt taggtgacac tata
//
