LOCUS       sequence-232502-        3635 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with sfGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3635)
  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 3635)
  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-06
FEATURES             Location/Qualifiers
     source          1..3635
                     /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"
     polyA_signal    801..922
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(838..857)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     892..911
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(929..962)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        969..1217
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     969..989
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1140..1159
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1219..1252)
                     /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(1289..1308)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1290..1308)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1395..1412)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1566..2154)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1646..1665)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2325..3116,3117..3185))
                     /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     2948..2967
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3186..3290)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3358..3376
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3414..3436)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3536..3555
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc agcaagggcg aggagctgtt caccggcgtg gtgcccatcc
      121 tggtggagct ggacggcgac gtgaacggcc acaagttcag cgtgcgcggc gagggcgagg
      181 gcgacgccac caacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      241 tgccctggcc caccctggtg accaccctga cctacggcgt gcagtgcttc agccgctacc
      301 ccgaccacat gaagcgccac gacttcttca agagcgccat gcccgagggc tacgtgcagg
      361 agcgcaccat cagcttcaag gacgacggca cctacaagac ccgcgccgag gtgaagttcg
      421 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
      481 acatcctggg ccacaagctg gagtacaact tcaacagcca caacgtgtac atcaccgccg
      541 acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacgtg gaggacggca
      601 gcgtgcagct ggccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
      661 tgcccgacaa ccactacctg agcacccaga gccgcctgag caaggacccc aacgagaagc
      721 gcgaccacat ggtgctgctg gagttcgtga ccgccgccgg catcacccac ggcatggacg
      781 agctgtacaa gtgaactagt aacttgttta ttgcagctta taatggttac aaataaagca
      841 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
      901 ccaaactcat caatgtatct tagtcgatat aacttcgtat agcatacatt atacgaagtt
      961 atgtcgagga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     1021 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     1081 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     1141 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     1201 ggaaaggacg aaacaccgat aacttcgtat agcatacatt atacgaagtt atggtaccga
     1261 tgcagctagc ccgcggatct gccggtctcc ctatagtgag tcgtattaat ttcgataagc
     1321 caggttaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     1381 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     1441 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     1501 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     1561 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     1621 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     1681 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     1741 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     1801 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     1861 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     1921 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     1981 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     2041 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2101 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     2161 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     2221 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     2281 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     2341 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2401 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     2461 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     2521 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     2581 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     2641 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     2701 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     2761 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     2821 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     2881 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     2941 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3001 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3061 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     3121 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     3181 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     3241 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     3301 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     3361 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     3421 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     3481 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     3541 tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac
     3601 aattaataca taaccttatg tatcatacac atacg
//
