LOCUS       sequence-232504-        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"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(123..144)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     731..752
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    771..892
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(808..827)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     862..881
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(899..932)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        939..1187
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     939..959
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1110..1129
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1189..1222)
                     /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(1259..1278)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1260..1278)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1365..1382)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1536..2124)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1616..1635)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2295..3086,3087..3155))
                     /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     2918..2937
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3156..3260)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3328..3346
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3384..3406)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3506..3525
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      121 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtccgc ggcgagggcg
      181 agggcgatgc caccaacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
      241 ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cgtggcctgc ttcagccgct
      301 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
      361 aggagcgcac catctctttc aaggacgacg gtacctacaa gacccgcgcc gaggtgaagt
      421 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
      481 gcaacatcct ggggcacaag ctggagtaca acttcaacag ccactacgtc tatatcacgg
      541 ccgacaagca gaagaactgc atcaaggcta acttcaagat ccgccacaac gttgaggacg
      601 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
      661 tgctgcccga caaccactac ctgagccatc agtccaagct gagcaaagac cccaacgaga
      721 agcgcgatca catggtcctg ctggagttcg tgaccgccgc ctgaactagt aacttgttta
      781 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
      841 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tagtcgatat
      901 aacttcgtat agcatacatt atacgaagtt atgtcgagga gggcctattt cccatgattc
      961 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     1021 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     1081 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     1141 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgat aacttcgtat
     1201 agcatacatt atacgaagtt atggtaccga tgcagctagc ccgcggatct gccggtctcc
     1261 ctatagtgag tcgtattaat ttcgataagc caggttaacc tgcattaatg aatcggccaa
     1321 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     1381 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     1441 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     1501 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     1561 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     1621 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     1681 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     1741 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     1801 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     1861 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     1921 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     1981 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2041 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2101 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2161 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2221 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     2281 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     2341 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     2401 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     2461 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     2521 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     2581 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     2641 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     2701 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     2761 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     2821 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     2881 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     2941 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3001 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3061 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3121 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     3181 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3241 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     3301 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     3361 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     3421 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     3481 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     3541 atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac
     3601 atacg
//
