LOCUS       sequence-227998-        3605 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with mClover3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3605)
  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 3605)
  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-08
FEATURES             Location/Qualifiers
     source          1..3605
                     /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..1170
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     922..942
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1093..1112
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1172..1205)
                     /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(1242..1261)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1243..1261)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1348..1365)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1519..2107)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1599..1618)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2278..3069,3070..3138))
                     /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     2901..2920
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3139..3243)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3311..3329
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3367..3389)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3489..3508
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3589..3605,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3589..3605,1)
                     /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 ggagggccta tttcccatga ttccttcata tttgcatata
      961 cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta
     1021 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     1081 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     1141 ttatatatct tgtggaaagg acgaaacacc gataacttcg tatagcatac attatacgaa
     1201 gttatggtac cgatgcagct agcccgcgga tctgccggtc tccctatagt gagtcgtatt
     1261 aatttcgata agccaggtta acctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     1321 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     1381 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     1441 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     1501 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     1561 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     1621 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     1681 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc
     1741 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     1801 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     1861 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     1921 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     1981 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     2041 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     2101 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     2161 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     2221 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     2281 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     2341 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     2401 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     2461 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     2521 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     2581 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     2641 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     2701 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     2761 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     2821 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     2881 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     2941 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     3001 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     3061 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     3121 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     3181 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     3241 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     3301 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
     3361 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     3421 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
     3481 taactatgcg gcatcagagc agattgtact gagagtgcac catatggaca tattgtcgtt
     3541 agaacgcggc tacaattaat acataacctt atgtatcata cacatacgat ttaggtgaca
     3601 ctata
//
